Mutant forms of cholera holotoxin as an adjuvant

ABSTRACT

Mutant cholera holotoxins having single or double amino acid substitutions or insertions have reduced toxicity compared to the wild-type cholera holotoxin. The mutant cholera holotoxins are useful as adjuvants in antigenic compositions to enhance the immune response in a vertebrate host to a selected antigen from a pathogenic bacterium, virus, fungus, or parasite, a cancer cell, a tumor cell, an allergen, or a self-molecule.

CROSS-REFERENCE TO OTHER APPLICATIONS

[0001] This application claims the benefit of the priority of U.S.provisional patent application No. 60/296,531, filed Jun. 7, 2001.

BACKGROUND OF THE INVENTION

[0002] The body's immune system activates a variety of mechanisms forattacking pathogens (Janeway, Jr, C A. and Travers P., eds., inImmunobiology, “The Immune System in Health and Disease,” SecondEdition, Current Biology Ltd., London, Great Britain (1996)). However,not all of these mechanisms are necessarily activated afterimmunization. Protective immunity induced by immunization is dependenton the capacity of an immunogenic composition to, elicit the appropriateimmune response to resist or eliminate the pathogen. Depending on thepathogen, this may require a cell-mediated and/or humoral immuneresponse.

[0003] Many antigens are poorly immunogenic or non-immunogenic whenadministered by themselves. Strong adaptive immune responses to antigensalmost always require that the antigens be administered together with anadjuvant, a substance that enhances the immune response (Audbert, F. M.and Lise, L. D. 1993 Immunology Today, 14: 281-284).

[0004] The need for effective immunization procedures is particularlyacute with respect to infectious organisms that cause acute infectionsat, or gain entrance to the body through, the gastrointestinal,pulmonary, nasopharyngeal or genitourinary surfaces. These areas arebathed in mucus, which contains immunoglobulins consisting largely ofsecretoty immunoglobulin IgA (Hanson, L. A., 1961 Intl. Arch. Allergy.Appl. Immunol., 18, 241-267; Tomasi, T. B., and Zigelbaum, S., 1963 J.Clin. Invest., 42, 1552-1560; and Tomasi, T. B., et al., 1965 J. Exptl.Med., 121, 101-124). This immunoglobulin is derived from large numbersof IgA-producing plasma cells, which infiltrate the lamina propriaregions underlying the mucosal membranes (Brandtzaeg, P., and Baklein,K, Scand. 1976 J. Gastroenterol., 11 (Suppl. 36), 1-45; and Brandtzaeg,P., 1984 “Immune Functions of Human Nasal Mucosa and Tonsils in Healthand Disease”, page 28 et seq. in Immunology of the Lung and UpperRespiratory Tract, Bienenstock, J., ed., McGraw-Hill, New York, N.Y.).The secretory immunoglobulin IgA is specifically transported to thelumninal surface through the action of the secretory component (Solari,R, and Kraehenbuhl, J-P, 1985 Immunol. Today, 6, 17-20).

[0005] Parenteral immunization regimens are usually ineffective ininducing secretory IgA responses. Secretory immunity is most oftenachieved through the direct immunization of mucosally associatedlymphoid tissues. Following their induction at one mucosal site, theprecursors of IgA-producing plasma cells extravasate and disseminate todiverse mucosal tissues where final differentiation to high-rate IgAsynthesis occurs (Crabbe, P. A, et al., 1969 J. Exptl. Med., 130,723-744; Bazin, H., et al., 1970 J. Immunol., 105, 1049-1051; Craig, S.W., and Cebra, J. J., 1971 J. Exptl. Med., 134, 188-200). Extensivestudies have demonstrated the feasibility of mucosal immunization toinduce this common mucosal immune system (Mestecky, J., et al., 1978 J.Clin. Invest., 61, 731-737). With rare exceptions the large doses ofantigen required to achieve effective immunization have made thisapproach impractical for purified antigens.

[0006] Among the strategies investigated to overcome this problem is theuse of mucosal adjuvants. A number of adjuvants that enhance the immuneresponse of antigens are known in the prior art (Elson, C. O., andEalding, W., 1984 J. Immunol., 132, 2736-2741). These adjuvants, whenmixed with an antigen, render the antigen particulate, helping retainthe antigen in the body for longer periods of time, thereby promotingincreased macrophage uptake and enhancing immune response. However,untoward reactions elicited by many adjuvants or their ineffectivenessin inducing mucosal immunity have necessitated the development of betteradjuvants for delivery of immunogenic compositions. Unfortunately,adjuvant development to date has been largely an empirical exercise(Janeway, Jr., et al, cited above at pages 12-25 to 12-35). Thus, arational and a more direct approach is needed to develop effectiveadjuvants for delivery of antigenic compositions.

[0007] It has been reported that the toxin secreted by the Gram-negativebacterium Vibrio cholerae (V. cholerae), the causative agent of thegastrointestinal disease cholera, is extremely potent as an adjuvant.Cholera toxin (CT) has been reported as a 382 amino acid sequence (SEQID NO: 1) (Mekalanos, J. J., et al, 1983 Nature, 306, 551-557), whichhas an 18 amino acid signal (amino acids 1 to 18 of SEQ ID NO: 1). Thecholera toxin holotoxin molecule is a hexaheteromeric complex thatconsists of a single peptide subunit designated CT-A (SEQ ID NO: 2 oramino acids 19 to 258 of SEQ ID NO: 1), which is responsible for theenzymatic activity of the toxin, and five identical peptide subunits,each designated CT-B (each having a 21 amino acid signal (amino acids259 to 279 of SEQ ID NO:1), followed by the CT-B peptide subunit (aminoacids 280 to 382 of SEQ ID NO: 1)), which are involved in the binding ofthe toxin to the intestinal epithelial cells as well as other cellswhich contain ganglioside GM, on their surface (Gill, D. M., 1976Biochem., 15,1242-1248; Cuatrecasas, P., 1973 Biochem., 12, 3558-3566).CT produced by V. cholerae has the CT-A subunit proteolytically cleavedwithin the single disulfide-linked loop between the cysteines at aminoacid positions 187 and 199 of the mature CT-A (SEQ ID NO: 2). Thiscleavage produces an enzymatically active A1 polypeptide (Kassis, S., etal., 1982 J. Biol. Chem., 257, 12148-12152) and a smaller polypeptideA2, which links fragment A1 to the CT-B pentamer (Mekalanos, J. J., etal., 1979 J. Biol. Chem., 254, 5855-5861). Toxicity results when theenzymatically active fragment CT-A1, upon entry into enterocytes,ADP-ribosylates a regulatory G-protein (Gsα). This leads to constitutiveactivation of adenylate cyclase, increased intracellular concentrationof cAMP, and secretion of fluid and electrolytes into the lumen of thesmall intestine (Gill, D. M., and Meren, R, 1978 Proc. Natl. Acad. Sci.,USA, 75, 3050-3054), thereby causing toxicity. In vitro, ADP-ribosyltransferase activity of CT is stimulated by the presence of accessoryproteins called ARFs, small GTP-binding proteins known to be involved invesicle trafficking within the eukaryotic cell (Welsh, C. F., et al.,“ADP-Ribosylation Factors: A Family of Guanine Nucleotide-BindingProteins that Activate Cholera Toxin and Regulate Vesicular Transport”,pages 257-280 in Handbook of Natural Toxins: Bacterial Toxins andVirulence Factors in Disease Vol. 8 (Moss, J., et al., eds., MarcelDekker, Inc., New York, N.Y. 1995).

[0008] Co-administration of CT with an unrelated antigen has beenreported to result in the induction of concurrent circulating andmucosal antibody responses to that antigen (Mekalanos, J. J., et al.,1983 Nature, 306, 551-557). To minimize the occurrence of undesirablesymptoms such as diarrhea caused by wild-type CT in humans, it would bepreferable to use as an adjuvant a form of the CT holotoxin that hassubstantially reduced toxicity. Mutants of CT have been suggested as ameans for achieving a more useful adjuvant. One way to rationally designmutant cholera toxin holotoxins (designated CT-CRMs) with substantiallyreduced toxicity is to identify and alter amino acid residues in thetoxin molecule that are completely conserved in the family of cholera(CT) and related heat-labile enterotoxins (LT-I, LT-IIa and LT-IIb) ofE. coli. Another rational way to generate mutant CT-CRMs withsubstantially reduced toxicity is to alter amino acid residues in theholotoxin molecule that have been identified as being important forNAD-binding based on the structural alignment of the CT backbone withthe backbone of related toxins possessing ADP-ribosyl transferase enzymeactivity such as diphtheria toxin (DT) and pertussis toxin (PT) (Holmes,R K, “Heat-labile enterotoxins (Escherichia coli)” in Guidebook toProtein Toxins and their Use in Cell Biology, Montecucco, C. andRappnoli, R., Eds., Oxford Univ. Press, Oxford, England (1997); andHolmes, R. K et al, “Cholera toxins and related enterotoxins ofGram-negative bacteria”, pp. 225-256 in Handbook of Natural Toxins:Bacterial Toxins and Virulence Factors in Disease, vol. 8, Moss. J., etal, Eds., Marcel Dekker, Inc., New York, N.Y. 1995).

[0009] Recently, one such rationally-designed, genetically-detoxifiedmutant of CT was disclosed wherein a single nonconservative amino acidsubstitution (glutamic acid to histidine) was introduced by altering theamino acid at position 29 in the mature A subunit (designatedCT-CRM_(E29H)). The resulting mutant cholera holotoxin demonstratedsubstantially reduced enzymatic toxicity, but with superior adjuvantingand immunogenic properties (International Patent Publication No. WO00/18434, incorporated in its entirety by reference).

[0010] Thus, there is a need to identify and/or rationally designadditional mutant forms of the CT holotoxin that have substantiallyreduced toxicity, yet possess the same or enhanced adjuvantingproperties as the wild-type CT holotoxin.

SUMMARY OF THE INVENTION

[0011] In one aspect, this invention provides novel mutant, immunogenicforms of cholera holotoxin (designated CT-CRMs) having significantlyreduced toxicity compared to wild-type cholera holotoxin (CT), but whichretain the ability to function as powerful stimulators of the immunesystem. Specifically, the invention pertains to five mutant choleraholotoxins (CT-CRMs), desirably generated by site-directed mutagenesisand having substantially reduced toxicity compared to wild-type CT, butwith no loss in adjuvanting properties.

[0012] In one embodiment, a novel CT-CRM of this invention comprises theamino acid sequence of CT subunit A or a fragment thereof, wherein theamino acid residue in the amino acid position 25 of the A subunit issubstituted with another amino acid, which substitution results in asubstantial reduction in toxicity. In a preferred embodiment of theinvention, the amino acid arginine at amino acid position 25 of the Asubunit is substituted with a tryptophan or a glycine. For determinationof the amino acid position, the sequence of CT-A is exemplified in SEQID NO: 2. However, other variants and fragments of CT-A may also beemployed.

[0013] In another embodiment, a novel immunogenic mutant CT-CRM of thisinvention comprises the amino acid sequence of CT subunit A or afragment thereof, wherein there is an insertion of a single amino acidresidue in the amino acid position 49 of the A subunit, which insertionresults in a substantial reduction in toxicity. In this aspect andthroughout this application, whenever it is stated that “there is aninsertion of a single (or multiple) amino acid residue(s) in the Asubunit”, this means that the wild-type residue(s) in amino acidposition(s) [insert amino acid number(s)] is (are) shifted downstream.In a preferred embodiment of the invention, the amino acid residuehistidine is inserted in the amino acid position 49 of the A subunit,thereby shifting the amino acid residues originally located at positions49, 50, etc., to positions 50, 51, etc.

[0014] In a third embodiment, a novel immunogenic, mutant CT-CRM of thisinvention has substantially reduced CT toxicity and comprises the aminoacid sequence of subunit A of CT or a fragment thereof, wherein there isan insertion of two amino acid residues in the amino acid positions 35and 36 in the A subunit, which insertion results in a substantialreduction in toxicity. In a preferred embodiment of this aspect of theinvention, the amino acid residues glycine and proline are inserted atthe amino acid positions 35 and 36 in the A subunit, thereby shiftingthe original amino acid residues at positions 35 and 36 to positions 37and 38, etc.

[0015] In yet another embodiment, a novel immunogenic, mutant CT-CRM ofthis invention has substantially reduced CT toxicity and comprises theamino acid sequence of subunit A of CT or a fragment thereof, whereinthere is an amino acid substitution in the amino acid residue 30 of theA subunit and an insertion of two amino acid residues in the amino acidpositions 31 and 32 in the A subunit, which substitution and insertionresults in a substantial reduction in toxicity. In a preferredembodiment of this aspect of the invention, the amino acid tryptophan issubstituted for tyrosine at amino acid position 30 of the A subunit, andthe amino acid residues alanine and histidine are inserted in the aminoacid positions 31 and 32, respectively, in the A subunit, therebyshifting the original amino acid residues at positions 31 and 32 topositions 33 and 34, etc.

[0016] In another aspect, the invention provides a method for producingthe novel CT-CRMs described above by employing site-directed mutagenesisof the DNA encoding the A subunit in the wild-type CT using conventionaltechniques, such that the mutagenized CT now has substantially reducedtoxicity without compromising the toxin's ability to stimulate an immuneresponse.

[0017] In yet another aspect of the invention, there is provided animmunogenic composition comprising a selected antigen, a mutant CT-CRMas described above as an adjuvant to enhance the immune response in avertebrate host to the antigen, and a pharmaceutically acceptablediluent, excipient or carrier. Preferably, the CT-CRM is useful for thegeneration or enhancement of systemic and/or mucosal antigenic immuneresponses in a vertebrate host to the selected antigen. The selectedantigen may be a polypeptide, peptide or fragment derived from apathogenic virus, bacterium, fungus or parasite. The selected antigenmay be a polypeptide, peptide or fragment derived from a cancer cell ortumor cell. The selected antigen may be a polypeptide, peptide orfragment derived from an allergen so as to interfere with the productionof IgE so as to moderate allergic responses to the allergen. Theselected antigen may be a polypeptide, peptide or fragment derived froma molecular portion thereof which represents those produced by a host (aself molecule) in an undesired manner, amount or location, such as thosefrom amyloid precursor protein so as to prevent or treat diseasecharacterized by amyloid deposition in a vertebrate host.

[0018] In one embodiment of this aspect of the invention, there isprovided an immunogenic composition selected comprising a selectedantigen as described above with a mutant, immunogenic CT-CRM protein ofthe invention, and a pharmaceutically acceptable diluent, excipient orcarrier.

[0019] In still another aspect, this invention provides a method forusing these CT-CRMs as adjuvants in immunogenic compositions or methodsfor increasing the ability of an antigenic composition containing aselected antigen as described above to elicit an immune response invertebrate host by including an effective adjuvanting amount of one ormore of the novel detoxified mutant cholera holotoxins (CT-CRMs)described above.

[0020] In yet a further aspect of the invention, there are provided DNAsequences encoding the novel immunogenic, mutant CT-CRMs withsubstantially reduced toxicity as described above. Preferably, the DNAsequence(s) encodes for both the mutant A subunit with reduced toxicityand subunit B. Alternatively, the DNA sequence may encode only themutant A subunit with reduced toxicity, where the altered or mutant CT-Ais fused with an additional binding domain, or is co-expressed with LT-Band allowed to co-assemble.

[0021] In a further aspect of the invention, there is provided a plasmidcontaining isolated and purified DNA sequence comprising a DNA sequenceencoding an immunogenic, detoxified, mutant cholera holotoxin asdescribed herein, and wherein such a DNA sequence is operatively linkedto regulatory sequences which direct expression of the CT-CRM in a hostcell. Preferably the regulatory sequences comprise an arabinoseinducible promoter. In one embodiment of this aspect, the inventionrelates to a plasmid, designated pLP915, that contains an isolated andpurified DNA sequence comprising a DNA sequence encoding an immunogenicmutant CT-CRM with substantially reduced toxicity wherein the amino acidarginine in amino acid position 25 of the A subunit is substituted withan tryptophan. In another embodiment of the invention, the inventionrelates to a plasmid, designated pLP911, that contains an isolated andpurified DNA sequence comprising a DNA sequence encoding an immunogenicmutant CT-CRM with substantially reduced toxicity wherein the amino acidarginine in the amino acid position 25 of the A subunit is substitutedwith a glycine.

[0022] In yet another embodiment of this aspect of the invention, thereis provided a plasmid, designated pLP907, that contains an isolated andpurified DNA sequence comprising a DNA sequence encoding an immunogenicmutant CT-CRM with substantially reduced toxicity wherein the amino acidresidue histidine is inserted in the amino acid position 49 in the Asubunit. In still another embodiment of this aspect, the inventionrelates to a plasmid, designated pLP909, that contains an isolated andpurified DNA sequence comprising a DNA sequence encoding an immunogenic,mutant CT-CRM with substantially reduced toxicity wherein the amino acidresidues glycine and proline are inserted in the amino acid positions 35and 36 in the A subunit. In still a further embodiment, the inventionrelates to a plasmid, designated pLP910, that contains an isolated andpurified DNA sequence comprising a DNA sequence encoding an immunogenic,mutant CT-CRM with substantially reduced toxicity wherein the amino acidresidue tyrosine in amino acid position 30 of the A subunit issubstituted with the amino acid residue tryptophan, and the amino acidresidues alanine and histidine are inserted in the amino acid positions31 and 32 in the A subunit.

[0023] In a further aspect of the invention, there is provided asuitable host cell line transformed, infected, transduced or transfectedwith a plasmid as described herein. The immunogenic, detoxified, mutantcholera holotoxins are produced by transforming, infecting, transducingor transfecting a suitable host cell with one of the plasmids describedabove and culturing the host cell under culture conditions which permitthe expression by the host cell of said recombinant immunogenic, mutantcholera holotoxin protein with substantially reduced toxicity.

[0024] These and other aspects of the invention will be apparent to oneof skill in the art upon reading of the following detailed descriptionof the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0025] Mutant forms of cholera holotoxin that exhibit reduced toxicity,but which retain their superior adjuvanting properties, and the utilityof these mutant forms of CTs as adjuvants in immunogenic compositionsare described herein.

[0026] A. Mutant, Detoxified Cholera Toxin Holotoxins

[0027] Novel mutant, detoxified immunogenic forms of cholera holotoxin(CT-CRMs) of this invention are characterized by significantly reducedtoxicity compared to a wild-type CT. However, such CT-CRMs retain theirability as powerful stimulators of the immune system. The CT-CRMs ofthis invention are characterized by one or several amino acidsubstitutions and/or insertions in the mature CT-A subunit of choleratoxin. The various mutant CT-A subunits of this invention also retainedtheir ability to assemble with CT-B subunits to form mutant CTholotoxins that resembled wild-type CT in adjuvanticity, but whichexhibited substantially reduced toxicity compared to the wild-type CT.The CT-CRMs of this invention may employ mutant or altered CT-A subunitsassociated with wild-type CT-B subunits to create a functionalholotoxin. Alternatively, the CT-CRMs of this invention may comprise thealtered or mutated CT-A subunits associated with altered or mutated CT-Bsubunits.

[0028] For determination of the amino acid position numbers describingthe locations of the amino acid substitutions or insertions in theCT-CRMs of this invention, the sequence of mature CT-A is exemplified asSEQ ID NO: 2, i.e., amino acids 19-258 of SEQ ID NO: 1, a wild-type CTsequence. The nucleotide sequence encoding the A subunit of the choleraholotoxin is set forth in International patent publication No. WO93/13202. Similarly, a suitable mature CT-B sequence may be illustratedby amino acids 280-382 of SEQ ID NO: 1. However, other variants,biotypes and fragments of CT-A and CT-B of V. cholerae may also beemployed as sequences containing the amino acid substitutions andinsertions described herein. See, for example, the ELTOR biotype of C.Shi et al, 1993 Sheng Wu Hua Hsueh Tsa Chih, 9(4):395-399; NCBI databaselocus No. AAC34728, and other sources of variants of V. cholerae toxin.

[0029] In one embodiment of this invention, the amino acid substitutionsor insertions resulting in some of the CT-CRMs of this invention are theresult of replacing one amino acid with another amino acid havingsimilar structural and/or chemical properties, i.e. conservative aminoacid replacements. “Conservative” amino acid substitutions or insertionsmay be made on the basis of similarity in polarity, charge, solubilityhydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues involved. For example, non-polar (hydrophobic) amino acidsinclude alanine, leucine, isoleucine, valine, proline, tryptophan, andmethionine; polar/neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; positivelycharged (basic) amino acids include arginine, lysine, and histidine; andnegatively charged (acidic) amino acids include aspartic acid andglutamic acid. This invention is exemplified by CT-CRMs, two bearing asingle amino acid substitution, one bearing a single amino acidinsertion, one bearing a double amino acid insertion, and one bearing asingle amino acid substitution and a double amino acid insertion. TheseCT-CRMs were generated as described in detail in Example 1 with thefollowing mutations in the A subunit as set forth in Table 1. TABLE 1Single and Double CT-CRM Mutants Amino Acid Substitution Native MutantAbbreviation 25 Arginine Tryptophan CT-CRM_(R25W) 25 Arginine GlycineCT-CRM_(R25G) 48 and 49 Threonine₄₈ Threonine₄₈, CT-CRM_(T48TH)Histidine₄₉ 34, 35, 36 Glycine₃₄ Glycine₃₄, CT-CRM_(G34GGP) Glycine₃₅,Proline₃₆ 30, 31, 32 Tyrosine₃₀ Tryptophan₃₀, CT-CRM_(Y30WAH) Alanine₃₁,Histidine₃₂

[0030] Thus, in one embodiment, a novel CT-CRM of this inventioncomprises the amino acid sequence of CT subunit A or a fragment thereof,wherein the amino acid residue in the amino acid position 25 of the Asubunit is substituted with another amino acid which substitutionresults in a substantial reduction in toxicity. In a preferredembodiment of the invention, the amino acid arginine at amino acidposition 25 of the A subunit is substituted with a tryptophan. Inanother preferred embodiment of the invention, the amino acid arginineat amino acid position 25 of the A subunit is substituted with aglycine. The resulting CT-CRM_(R25W) and CT-CRM_(R25G) each demonstratesuperior adjuvanting properties.

[0031] A novel CT-CRM of this invention comprises a single amino acidinsertion at the amino acid position at the amino acid position adjacentto the amino acid residue at the amino acid position 48 in the Asubunit, which insertion results in a substantial reduction of toxicity.In a preferred embodiment of the invention the amino acid histidine isinserted adjacent to the amino acid position 48 in the A subunit,resulting in the mutant CT-CRM_(T48TH), which demonstrates superioradjuvanting properties.

[0032] Another novel CT-CRM of this invention comprises a double aminoacid insertion in the amino acid positions 35 and 36 adjacent to theamino acid residue at the amino acid position 34, in the A subunit,which insertion results in a substantial reduction of toxicity. In apreferred embodiment of the invention the amino acids glycine andproline are inserted adjacent to the amino acid position glycine 34 inthe A subunit, resulting in the mutant CT-CRM_(G34GGP), whichdemonstrates superior adjuvanting properties.

[0033] Yet another novel CT-CRM of this invention comprises a singleamino acid substitution at the amino acid position 30 and double aminoacid insertion at the amino acid positions 31 and 32 adjacent to theamino acid residue at the amino acid position 30), in the A subunit,which substitution and insertion results in a substantial reduction oftoxicity. In a preferred embodiment of the invention, the amino acidresidue tyrosine at amino acid position 30 is substituted with the aminoacid residue tryptophan and the amino acid residues alanine andhistidine are inserted thereafter, resulting in the mutantCT-CRM_(Y30WAH), which demonstrates superior adjuvanting properties.

[0034] Still other CT-CRMs of this invention may contain at least thesingle substitutions or single or double mutations describedspecifically above and at least one additional mutation at a positionother than at one or more of the amino acid residues 25, 30, 31, 32, 34,35, 36, 48 and 49, as set forth above. International patent publicationNo. WO 93/13202, which is hereby incorporated by reference, describes aseries of mutations in the CT-A subunit that serve to reduce thetoxicity of the cholera holotoxin. These mutations include makingsubstitutions for the arginine at amino acid 7, the aspartic acid atposition 9, the arginine at position 11, the glutamic acid at position29, the histidine at position 44, the valine at position 53, thearginine at position 54, the serine at position 61, the serine atposition 63, the histidine at position 70, the valine at position 97,the tyrosine at position 104, the proline at position 106, the histidineat position 107, the glutamic acid at position 110, the glutamic acid atposition 112, the serine at position 114, the tryptophan at position127, the arginine at position 146 and the arginine at position 192.International patent publication No. WO 98/42375, which is herebyincorporated by reference, describes making a substitution for theserine at amino acid 109 in the A subunit, which serves to reduce thetoxicity of the cholera holotoxin.

[0035] Other useful CT-CRM mutant proteins useful in this inventioninclude a full-length holotoxin with one or more of the specificmutations provided above, and a hexameric, CT-CRM polypeptide or afragment thereof containing the mutagenized residues described above andwhich protein, polypeptide or fragment retains the adjuvanticity ofwild-type CT from which it is derived, but is characterized by reducedtoxicity. Immunologically active fragments of these CT-CRMs with reducedenzymatic activity may also be useful in the methods and compositions ofthis invention. Fragments ordinarily will contain at least at leastabout 25 contiguous amino acids of the CT-CRM subunit proteinscontaining the sites of mutagenesis noted above. More typically a CT-CRMsubunit fragment contains at least about 75 contiguous amino acids ofthe A or B subunits. Another fragment of a CT-CRM subunit contains atleast about 100 contiguous amino acids of either subunit. Still anotherembodiment of a CT-CRM CT-A subunit may contain about 150 amino acids orless than 240 amino acids.

[0036] A fragment of the CT-CRMs described herein is useful in themethods and compositions described below if it generates or enhances theimmune response to selected antigens in the vertebrate host. Fragmentsinclude truncations of the carboxy-terminal region of the CT-CRMsubunits. For example, a CT-CRM truncated so that it contains only aCT-A mutant subunit is a desirable fragment. Similarly, CT-A subunitstruncated at about residues 240 or 250 are desirable fragments. Stillother fragments CT-CRMs of this invention may be selected. Additionalfragments of the CT-CRM holotoxin may contain less than five repetitionsof the CT-B subunits or truncated CT-B subunits. The foregoing fragmentsmay also contain one or more of the specific mutations described above.

[0037] Other suitable CT-CRM proteins may include those in which one ormore of the amino acid residues includes a substituted group. Stillanother suitable CT-CRM holotoxin protein is one in which one or more ofthe subunits of the hexameric CT-CRM protein is fused with anothercompound, such as a compound to increase the half-life of the molecule(for example, polyethylene glycol). Another suitable CT-CRM protein isone in which additional amino acids are fused to one or more of thepolypeptide subunits, such as a leader or secretory sequence, or asequence which is employed to enhance the immunogenicity of the CT-CRMprotein. Still other modifications of the CT-CRMs include theabove-mentioned deletion of the CT-A signal or leader sequences at the Nterminus of CT, i.e., amino acids 1-18 of SEQ ID NO: 1, and/or thedeletion of the CT-B signal or leader sequence, i.e., at amino acids259-279 of SEQ ID NO: 1, and/or the deletion of other regions that donot effect immunogenicity. Similarly, a modification of the CT-CRMsdescribed herein includes include replacing either signal or leadersequences with other signal or leader sequences. See, e.g., U.S. Pat.No. 5,780,601, incorporated by reference herein.

[0038] Still another example of suitable CT-CRM proteins are those inwhich optional amino acids (e.g., -Gly-Ser-) or other amino acid orchemical compound spacers may be included at the termini of thepolypeptide subunits for the purpose of lining multiple holotoxinproteins together or to a carrier. For example, useful CT-CRMs mayinclude one or more of the above-described CT-CRMs or subunits thereofcoupled to a carrier protein. Alternatively, a useful CT-CRM may bepresent in a fusion protein containing multiple CT-CRMs, optionallycoupled to carrier protein.

[0039] For these embodiments, the carrier protein is desirably a proteinor other molecule that can enhance the immunogenicity of the selectedCT-CRM. Such a carrier may be a larger molecule that also has anadjuvanting effect. Exemplary conventional protein carriers include,without limitation, E. coli DnaK protein, galactokinase (GalK, whichcatalyzes the first step of galactose metabolism in bacteria),ubiquitin, α-mating factor, β-galactosidase, and influenza NS-1 protein.Toxoids (i.e., the sequence which encodes the naturally occurring toxin,with sufficient modifications to eliminate its toxic activity) such asdiphtheria toxoid and tetanus toxoid, their respective toxins, and anymutant forms of these proteins, such as CRM₁₉₇ (a non-toxic form ofdiphtheria toxin, see U.S. Pat. No. 5,614,382), may also be employed ascarriers. Other carriers include exotoxin A of Pseudomonas aeruginosa,heat labile toxins of E. coli and rotaviral particles (includingrotavirus and VP6 particles). Alternatively, a fragment or epitope ofthe carrier protein or other immunogenic protein may be used. Forexample, a hapten may be coupled to a T cell epitope of a bacterialtoxin. See U.S. Pat. No. 5,785,973. Similarly a variety of bacterialheat shock proteins, e.g., mycobacterial hsp-70 may be used.Glutathione-S-transferase (GST) is another useful carrier. One of skillin the art can readily select an appropriate carrier for use in thiscontext. The fusion proteins may be formed by standard techniques forcoupling proteinaceous materials. Fusions may be expressed from fusedgene constructs prepared by recombinant DNA techniques as describedbelow.

[0040] Other suitable CT-CRMs described herein can differ from thespecifically exemplified CT-CRMs by modifications that do not reviveenzymatic toxicity, and do not diminish adjuventicity, or bycombinations of such attributes. Preferably, the amino acidsubstitutions are the result of replacing one amino acid with anotheramino acid having similar structural and/or chemical properties, i.e.conservative amino acid replacements. “Conservative” amino acidsubstitutions may be made on the basis of similarity in polarity,charge, solubility hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved. For example, non-polar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, tryptophan, and methionine; polar/neutral amino acids includeglycine, serine, threonine, cysteine, tyrosine, asparagine, andglutamine; positively charged (basic) amino acids include arginine,lysine, and histidine; and negatively charged (acidic) amino acidsinclude aspartic acid and glutamic acid.

[0041] For example, conservative amino acid changes may be made, which,although they alter the primary sequence of the subunits of the CT-CRMprotein, do not normally alter the function of the molecule. In makingsuch changes, the hydropathic index of amino acids can be considered.The importance of the hydropathic amino acid index in conferringinteractive biologic function on a polypeptide is generally understoodin the art (Kyte & Doolittle, 1982, J. Mol. Biol., 157(1):105-32). It isknown that certain amino acids can be substituted for other amino acidshaving a similar hydropathic index or score and still result in apolypeptide with similar biological activity. Each amino acid has beenassigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics. Those indices are: isoleucine (+4.5); valine(+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

[0042] It is believed that the relative hydropathic character of theamino acid residue determines the secondary and tertiary structure ofthe resultant polypeptide, which in turn defines the interaction of thepolypeptide with other molecules, such as enzymes, substrates,receptors, antibodies, antigens, and the like. It is known in the artthat an amino acid can be substituted by another amino acid having asimilar hydropathic index and still obtain a functionally equivalentpolypeptide. In such changes, the substitution of amino acids whosehydropathic indices are within +/−2 is preferred, those within +/−1 areparticularly preferred, and those within +/−0.5 are even moreparticularly preferred.

[0043] Substitution or insertion of like amino acids can also be made onthe basis of hydrophilicity, particularly where the biologicallyfunctional equivalent polypeptide or peptide thereby created is intendedfor use in immunological embodiments. U.S. Pat. No. 4,554,101,incorporated herein by reference, states that the greatest local averagehydrophilicity of a polypeptide, as governed by the hydrophilicity ofits adjacent amino acids, correlates with its immunogenicity andantigenicity, i.e. with a biological property of the polypeptide. Asdetailed in U.S. Pat. No. 4,554,101, the following hydrophilicity valueshave been assigned to amino acid residues: arginine (+3.0); lysine(+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); proline (−0.5±1);threonine (−0.4); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). It isunderstood that an amino acid can be substituted for another having asimilar hydrophilicity value and still obtain a biologically equivalent,and in particular, an immunologically equivalent polypeptide. In suchchanges, the substitution of amino acids whose hydrophilicity values arewithin ±2 is preferred; those within ±1 are particularly preferred; andthose within ±0.5 are even more particularly preferred.

[0044] As outlined above, amino acid substitutions are generally basedon the relative similarity of the amino acid side-chain substituents,for example, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions which take various of the foregoingcharacteristics into consideration are well known to those of skill inthe art and include: arginine and lysine; glutamate and aspartate;serine and threonine; glutamine and asparagine; and valine, leucine andisoleucine.

[0045] In addition, modifications, which do not normally alter theprimary sequence of the CT-CRM protein, include in vivo or in vitrochemical derivatization of polypeptides, e.g., acetylation, methylation,or carboxylation. Also included as CT-CRMs of this invention are theseproteins modified by glycosylation, e.g., those made by modifying theglycosylation patterns of a polypeptide during its synthesis andprocessing or in further processing steps; or by exposing thepolypeptide to enzymes which affect glycosylation, such as mammalianglycosylating or deglycosylating enzymes. Also embraced as CT-CRMs arethe above-identified mutagenized sequences, which have phosphorylatedamino acid residues, e.g., phosphotyrosine, phosphoserine, orphosphothreonine.

[0046] Also included as CT-CRMs of this invention are the abovesequences that have been modified using ordinary molecular biologicaltechniques so as to improve their resistance to proteolytic degradationor to optimize solubility properties. Among such CT-CRMs are includedthose containing residues other than naturally occurring L-amino acids,e.g., D-amino acids or non-naturally occurring synthetic amino acids.Among other known modifications which may be present in CT-CRMs of thepresent invention are, without limitation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cystine, formation of pyroglutamate, formylation,gamma-carboxylation, GPI anchor formation, hydroxylation, iodination,methylation, myristoylation, oxidation, proteolytic processing,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

[0047] The phenotypic effects of the novel CT-CRMs of Table 1 on thestructure and function of CT were assessed. The mutant A subunits witheither a single amino acid substitution, a single amino acid insertion,a double amino acid insertion or a single amino acid substitution anddouble amino acid insertion, generated by site directed mutagenesis ofthe CT-encoding gene were also able to assemble with CT-B subunits intoimmunoreactive holotoxin in the presence of subunit B as determined bynon-denaturing gel electrophoresis assay (see Table 2, Example 2). Eachmutant holotoxin was also tested in a Y-1 adrenal tumor cell assay todetermine its residual toxicity compared to wild-type CT holotoxin (seeTables 3 and 4, Example 3). These holotoxins resembled wild-type CT intheir adjuvanticities, but the results presented in Table 3 demonstratethat the mutant CT-CRMs had substantially reduced toxicity when comparedwith wild-type cholera holotoxin. The residual toxicity of the CT-CRMswith single and double amino acid substitutions were substantiallyreduced in comparison to that of the wild-type CT. These datademonstrate that the mutant CT-CRMs are holotoxins and are substantiallyless toxic than wild-type CT. Specifically, the mutant CT-CRMs displayedsignificantly lower levels of toxicity than the wild-type choleraholotoxin in the Y-1 mouse adrenal cell assay.

[0048] Each of the mutant CT-CRMs was also compared to wild-type CT inan ADP-ribosyltransferase activity assay (see Example 4). The results,which were generally in agreement with the toxicity data generated inthe Y-1 adrenal cell assay, indicated that the ADP-ribosyltransferaseactivity of the various CT-CRMs was substantially reduced when comparedto wild-type CT (Tables 5 and 6).

[0049] As used herein, the terms and phrases “the holotoxin has reducedtoxicity” or “substantially less toxic” or the like mean that the CT-CRMmutant of this invention, such as the five CT-CRM mutants describedherein (CT-CRM_(R25W), CT-CRM_(R25G), CT-CRM_(T48TH), CT-CRM_(G34GGP),CT-CRM_(Y30WAH)), exhibits a substantially lower toxicity per unit ofpurified toxin protein compared to the wild-type CT. This “reducedtoxicity” enables each mutant to be used as an adjuvant in animmunogenic composition without causing significant side effects,particularly those known to be associated with CT, e.g., diarrhea. Asdescribed in more detail below, the mutant CT-CRMs of this inventiondisplay significantly lower levels of toxicity than the wild-type CT inthe Y-1 mouse adrenal cell assay, and a significantly reducedADP-ribosyltransferase activity when compared to wild-type CT.

[0050] The immunogenic mutant CT-CRMs according to the present inventionexhibit a balance of reduced toxicity and retained adjuvanticity, suchthat the resulting mutant CT protein functions as an adjuvant whilebeing tolerated safely by the vertebrate host to which it is introduced.As indicated in the examples below, results in murine model assaysystems indicate that the mutant CT-CRMs disclosed herein were able tosignificantly augment mucosal and systemic immune responses followingintranasal administration of disparate antigens. Furthermore, even inthe presence of pre-existing anti-CT immune responses, the mutantCT-CRMs were able to serve as efficient mucosal adjuvants. The studiesthat support these characteristics of the CT-CRMs of this invention aresummarized below and more specifically stated in the Examples.

[0051] To evaluate the efficacy of the mutant CT-CRMs as mucosaladjuvants for compositions containing bacterial or viral antigens thathave been identified as candidates for inclusion in immunogeniccompositions, two disparate model antigen systems were examined: (1) therecombinant P4 outer membrane protein (also known as protein “e”(rP4))of the nontypable Haemophilus influenzae bacterium (NTHi), (see U.S.Pat. No. 5,601,831), and (2) the native UspA2 outer membrane protein ofthe Moraxella catarrhalis bacterium (International Patent PublicationNo. WO 98/28333).

[0052] Importantly, the data demonstrate that the mutant CT-CRMs areable to augment mucosal and systemic immune responses followingintranasal (IN) administration of disparate antigens. Results in murinemodel systems indicate that all mutant CT-CRMs disclosed herein wereable to significantly augment mucosal and systemic immune responsesfollowing intranasal administration of these disparate antigens.Furthermore, even in the presence of pre-existing anti-CT immuneresponses, the mutant CT-CRMs were able to serve as efficient mucosaladjuvants (see Tables 6-18).

[0053] The immunogenic mutant CT-CRMs according to the present inventionexhibit a balance of reduced toxicity and retained adjuvanticity, suchthat the protein functions as an adjuvant while being tolerated safelyby the vertebrate host immunized with the composition.

[0054] B. Nucleic Acid Molecules Encoding CT-CRMs

[0055] Another aspect of this invention includes isolated, synthetic orrecombinant nucleic acid molecules and sequences encoding theabove-described CT-CRMs and/or subunits thereof having the specifiedsite directed mutations, substitutions and/or insertions, or fragmentsthat may further contain one or more of those mutations, substitutionsand/or insertions.

[0056] An isolated nucleotide molecule comprising a nucleic acidsequence encoding a CT-CRM protein may be preferably under the controlof regulatory sequences that direct expression of the CT-CRM in a hostcell. As described herein, such nucleic acid molecules may be used toexpress the CT-CRM protein in vitro or to permit expression of theCT-CRM protein in vivo in a human.

[0057] As used herein, the term “isolated nucleotide molecule orsequence” refers to a nucleic acid segment or fragment which is freefrom contamination with, other biological components that may beassociated with the molecule or sequence in its natural environment. Forexample, one embodiment of an isolated nucleotide molecule or sequenceof this invention is a sequence separated from sequences which flank itin a naturally occurring state, e.g., a DNA fragment which has beenremoved from the sequences which are normally adjacent to the fragment,such as the sequences adjacent to the fragment in a genome in which itnaturally occurs. Further, the nucleotide sequences and molecules ofthis invention have been altered to encode a CT-CRM protein of thisinvention. Thus, the term “isolated nucleic acid molecule or sequence”also applies to nucleic acid sequences or molecules that have beensubstantially purified from other components that naturally accompanythe unmutagenized nucleic acid, e.g., RNA or DNA or proteins, in thecell. An isolated nucleotide molecule or sequence of this invention alsoencompasses sequences and molecules that have been prepared by otherconventional methods, such as recombinant methods, synthetic methods,e.g., mutagenesis, or combinations of such methods. The nucleotidesequences or molecules of this invention should not be construed asbeing limited solely to the specific nucleotide sequences presentedherein, but rather should be construed to include any and all nucleotidesequences which share homology (i.e., have sequence identity) with thenucleotide sequences presented herein.

[0058] The terms “substantial homology” or “substantial similarity,”when referring to a nucleic acid or fragment thereof, indicate that,when optimally aligned with appropriate nucleotide insertions ordeletions with another nucleic acid (or its complementary strand), thereis nucleotide sequence identity in at least about 70% of the nucleotidebases, as measured by any well-known algorithm of sequence identity,such as FASTA, a program in GCG Version 6.1. The term “homologous” asused herein, refers to the sequence similarity between two polymericmolecules, e.g., between two nucleic acid molecules, e.g., two DNAmolecules or two RNA molecules, or between two polypeptide molecules.When a nucleotide or amino acid position in both of the two molecules isoccupied by the same monomeric nucleotide or amino acid, e.g., if aposition in each of two DNA molecules is occupied by adenine, then theyare homologous at that position. The homology between two sequences is adirect function of the number of matching or homologous positions, e.g.,if half (e.g., five positions in a polymer ten subunits in length) ofthe positions in two compound sequences are homologous, then the twosequences are 50% homologous. If 90% of the positions, e.g., 9 of 10,are matched or homologous, the two sequences share 90% homology. By wayof example, the DNA sequences 3′ATTGCC5′ and 3′TATGCG5′ share 50%homology. By the term “substantially homologous” as used herein, ismeant DNA or RNA which is about 70% homologous, more preferably about80% homologous, and most preferably about 90% homologous to the desirednucleic acid.

[0059] The invention is also directed to an isolated nucleotide moleculecomprising a nucleic acid sequence that is at least 70%, 80% or 90%homologous to a nucleic acid sequence encoding a CT-CRM protein orsubunit of this invention that has reduced enzymatic toxicity comparedto wild-type CT protein and that retains adjuvanticity of the wild-typeCT. Furthermore, due to the degeneracy of the genetic code, anythree-nucleotide codon that encodes a mutant or substituted amino acidresidue of CT-CRM, described herein is within the scope of theinvention.

[0060] Where, as discussed herein, CT-CRMs, mutant CT-A subunits, ormutant CT-B subunits, and/or DNA sequences encoding them, or othersequences useful in nucleic acid molecules or compositions describedherein are defined by their percent homologies or identities toidentified sequences, the algorithms used to calculate the percenthomologies or percent identities include the following: theSmith-Waterman algorithm (J. F. Collins et al, 1988, Comput. Appl.Biosci., 4:67-72; J. F. Collins et al, Molecular Sequence Comparison andAlignment, (M. J. Bishop et al, eds.) In Practical Approach Series:Nucleic Acid and Protein Sequence Analysis XVIII, IRL Press: Oxford,England, UK (1987) pp.417), and the BLAST and FASTA programs (E. G.Shpaer et al, 1996, Genomics, 38:179-191). These references areincorporated herein by reference.

[0061] By describing two DNAs as being “operably linked” as used herein,is meant that a single-stranded or double-stranded DNA comprises each ofthe two DNAs and that the two DNAs are arranged within the DNA in such amanner that at least one of the DNA sequences is able to exert aphysiological effect by which it is characterized upon the other.

[0062] Preferably, for use in producing a CT-CRM protein of thisinvention or in administering it for in vivo production in a cell, eachCT-CRM protein encoding sequence and necessary regulatory sequences arepresent in a separate viral or non-viral recombinant vector (includingnon-viral methods of delivery of a nucleic acid molecule into a cell).Alternatively, two or more of these nucleic acid sequences encodingduplicate copies of a CT-CRM protein or encoding multiple differentCT-CRMs of this invention may be contained in a polycistronictranscript, i.e., a single molecule designed to express multiple geneproducts.

[0063] The invention further relates to vectors, particularly plasmids,containing isolated and purified DNA sequences comprising DNA sequencesthat encode an immunogenic mutant cholera holotoxin. Desirableembodiments include plasmids containing DNA sequences which encode, forexample, an immunogenic mutant cholera holotoxin having single aminoacid substitutions at amino acid residue 25 of CT-A, a single amino acidinsertion between amino acid residues 48 and 49 of CT-A, double aminoacid insertions between amino acid residues 34 and 35 of CT-A, or asingle amino acid substitution at amino acid residues 30 and a doubleamino acid insertion between amino acids 30 and 31 of CT-A By the term“vector” as used herein, is meant a DNA molecule derived from viral ornon-viral, e.g., bacterial, species that has been designed to encode anexogenous or heterologous nucleic acid sequence. Thus, the term includesconventional bacterial plasmids. Such plasmids or vectors can includeplasmid sequences from viruses or phages. Such vectors includechromosomal, episomal and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, bacteriophages, yeast episomes, yeastchromosomal elements, and viruses. Vectors may also be derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, cosmids, and phagemids. The term alsoincludes non-replicating viruses that transfer a gene from one cell toanother. The term should also be construed to include non-plasmid andnon-viral compounds which facilitate transfer of nucleic acid intocells, such as, for example, polylysine compounds and the like.

[0064] The nucleic acid molecules of the invention include non-viralvectors or methods for delivery of the sequences encoding the CT-CRMprotein to a host cell according to this invention. A variety ofnon-viral vectors are known in the art and may include, withoutlimitation, plasmids, bacterial vectors, bacteriophage vectors, “naked”DNA and DNA condensed with cationic lipids or polymers.

[0065] Examples of bacterial vectors include, but are not limited to,sequences derived from bacille Calmette Guérin (BCG), Salmonella,Shigella, E. coli, and Listeria, among others. Suitable plasmid vectorsinclude, for example, pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9,pUC18, pUC19, pLG339, pR290, pK37, pKC101, pAC105, pVA51, pKH47, pUB110,pMB9, pBR325, Col E1, pSC101, pBR313, pML21, RSF2124, pCR1, RP4, pBAD18,and pBR328.

[0066] Examples of suitable inducible Escherichia coli expressionvectors include pTrc (Amann et al., 1988 Gene, 69:301-315), thearabinose expression vectors (e.g., pBAD18, Guzman et al, 1995 J.Bacteriol., 177:4121-4130), and pETIId (Studier et al., 1990 Methods inEnzymology, 185:60-89). Target gene expression from the pTrc vectorrelies on host RNA polymerase transcription from a hybrid trp-lac fusionpromoter. Target gene expression from the pETIId vector relies ontranscription from a T7 gn10-lac fusion promoter mediated by acoexpressed viral RNA polymerase T7 gn 1. This viral polymerase issupplied by host strains BL21 (DE3) or HMS I 74(DE3) from a residentprophage harboring a T7 gn1 gene under the transcriptional control ofthe lacUV5 promoter. The pBAD system relies on the inducible arabinosepromoter that is regulated by the araC gene. The promoter is induced inthe presence of arabinose.

[0067] As one example, a plasmid, designated pLP9911, contains anisolated and purified DNA sequence comprising a DNA sequence encoding animmunogenic mutant CT-CRM with substantially reduced toxicity having asingle amino acid substitution (arginine to tryptophan) at amino acidposition 25 in the A subunit (CT-CRM_(R25W)). As another example, aplasmid, designated pLP9915, contains an isolated and purified DNAsequence comprising a DNA sequence encoding an immunogenic mutant CT-CRMwith substantially reduced toxicity having a single amino acidsubstitution (arginine to glycine) at amino acid position 25 in the Asubunit (CT-CRM_(R25G)). A third plasmid, designated pLP9907, containsan isolated and purified DNA sequence comprising a DNA sequence encodingan immunogenic mutant CT-CRM with substantially reduced toxicity whereina single amino acid histidine is inserted at the amino acid position 49adjacent to the amino acid residue threonine at the amino acid position48 in the A subunit (CT-CRM_(T48TH)). Another exemplary plasmid isdesignated pLP9909. This plasmid contains an isolated and purified DNAsequence comprising a DNA sequence encoding an immunogenic, mutantCT-CRM with substantially reduced toxicity wherein a double amino acidinsertion of amino acid residues glycine and proline is inserted in theamino acid positions 35 and 36 adjacent to the amino acid residueglycine at the amino acid position 34 in the A subunit(CT-CRM_(G34GGP)). Another plasmid exemplified in this invention isdesignated pLP9910. It contains an isolated and purified DNA sequencecomprising a DNA sequence encoding an immunogenic, mutant CT-CRM withsubstantially reduced toxicity wherein a single amino acid substitutionat the amino acid position 30 (substitution of the amino acid residuetyrosine at amino acid position 30 with amino acid residue tryptophan)and a double amino acid insertion of amino acid residues (alanine andhistidine) in the amino acid positions 31 and 32 adjacent to the aminoacid residue at the amino acid position 30 are made in the A subunit(CT-CRM_(Y30WAH)).

[0068] Another type of useful vector is a single or double-strandedbacteriophage vector. For example, a suitable cloning vector includes,but is not limited to the vectors such as bacteriophage λ vector system,λgt11, μgt μWES.tB, Charon 4, λgt-WES-λB, Charon 28, Charon 4A,λgt-1-λBC, λgt-1-λB, M13 mp7, M13 mp8, or M13 mp9, among others.

[0069] In another embodiment, the expression vector is a yeastexpression vector. Examples of vectors for expression in a yeast such asS. cerevisiae include pYepSec I (Baldari, et al., 1987 Protein Eng.,1(5):433-437), pMFa (Kurjan and Herskowitz, 1982 Cell, 30(3):933-943),pJRY88 (Schultz et al., 1987 Gene, 61(2): 123-133), and pYES2(Invitrogen Corporation, San Diego, Calif.).

[0070] Alternatively, baculovirus expression vectors are used.Baculovirus vectors available for expression of proteins in culturedinsect cells (e.g., Sf 9 or Sf 21 cells) include the pAc series (Smithet al., 1983 Biotechnol., 24:434-443) and the pVL series (Luckow andSummers, 1989 Virol., 170(1):31-39).

[0071] In yet another embodiment, a mammalian expression vector is usedfor expression in mammalian cells. Examples of mammalian expressionvectors include pCDM8 (Seed, 1987 Nature, 329:840-842) and pMT2PC(Kaufman et al., 1987 EMBO J., 6(1):187-93). When used in mammaliancells, the expression vector's control functions are often provided byviral regulatory elements.

[0072] One type of recombinant vector is a recombinant single ordouble-stranded RNA or DNA viral vector. A variety of viral vectorsystems are known in the art. Examples of such vectors include, withoutlimitation, recombinant adenoviral vectors, herpes simplex virus(HSV)-based vectors, adeno-associated viral (AAV) vectors, hybridadenoviral/AAV vectors, recombinant retroviruses or lentiviruses,recombinant poxvirus vectors, recombinant vaccinia virus vectors, SV-40vectors, insect viruses such as baculoviruses, and the like that areconstructed to carry or express a selected nucleic acid composition ofinterest.

[0073] Retrovirus vectors that can be employed include those describedin EP 0 415 731; International Patent Publication Nos. WO 90/07936; WO94/03622; WO 93/25698; and WO 93/25234; U.S. Pat. No. 5,219,740;International Patent Publication Nos. WO 93/11230 and WO 93/10218; Vileand Hart, 1993 Cancer Res. 53:3860-3864; Vile and Hart, 1993 Cancer Res.53:962-967; Ram et al., 1993 Cancer Res. 53:83-88; Takamiya et al., 1992J. Neurosci. Res. 33:493-503; Baba et al., 1993 J. Neurosurg.79:729-735; U.S. Pat. No. 4,777,127; GB Patent No. 2,200,651; and EP 0345 242. Examples of suitable recombinant retroviruses include thosedescribed in International Patent Publication No. WO 91/02805.

[0074] Alphavirus-based vectors may also be used as the nucleic acidmolecule encoding the CT-CRM protein. Such vectors can be constructedfrom a wide variety of alphaviruses, including, for example, Sindbisvirus vectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), RossRiver virus (ATCC VR-373; ATCC VR-1246) and Venezuelan equineencephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCCVR-532). Representative examples of such vector systems include thosedescribed in U.S. Pat. Nos. 5,091,309; 5,217,879; and 5,185,440; andInternational Patent Publication Nos. WO 92/10578; WO 94/21792; WO95/27069; WO 95/27044; and WO 95/07994.

[0075] Examples of adenoviral vectors include those described byBerkner, 1988 Biotechniques 6:616-627; Rosenfeld et al., 1991 Science252:431-434; International Patent Publication No. WO 93/19191; Kolls etal., 1994 PNAS 91:215-219; Kass-Eisler et al., 1993 PNAS 90:11498-11502;Guzman et al., 1993 Circulation 88:2838-2848; Guzman et al., 1993 Cir.Res. 73:1202-1207; Zabner et al., 1993 Cell 75:207-216; Li et al., 1993Hum. Gene Ther. 4:403-409; Cailaud et al., 1993 Eur. J. Neurosci.5:1287-1291; Vincent et al., 1993 Nat. Genet. 5:130-134; Jaffe et al.,1992 Nat. Genet. 1:372-378; and Levrero et al., 1991 Gene 101: 195-202.Exemplary adenoviral vectors include those described in InternationalPatent Publication Nos. WO 94/12649; WO 93/03769; WO 93/19191; WO94/28938; WO 95/11984 and WO 95/00655. Other adenoviral vectors includethose derived from chimpanzee adenoviruses, such as those described inU.S. Pat. No. 6,083,716.

[0076] Another viral vector is based on a parvovirus such as anadeno-associated virus (AAV). Representative examples include the AAVvectors described in International Patent Publication No. WO 93/09239,Samulski et al., 1989 J. Virol. 63:3822-3828; Mendelson et al., 1988Virol. 166:154-165; and Flotte et al., 1993 PNAS 90:10613-10617. Otherparticularly desirable AAV vectors include those based upon AAV1; see,International Patent Publication No. WO 00/28061, published May 18,2000. Other desirable AAV vectors include those which: are pseudotyped,i.e., contain a minigene composed of AAV 5′ ITRs, a transgene, and AAV3′ ITRs packaged in a capsid of an AAV serotype heterologous to the AAVITRs. Methods of producing such pseudotyped AAV vectors are described indetail in International Patent Publication No. WO01/83692.

[0077] In an embodiment in which the nucleic acid molecule of theinvention is “naked DNA”, it may be combined with polymers includingtraditional polymers and non-traditional polymers such ascyclodextrin-containing polymers and protective, interactivenoncondensing polymers, among others. The “naked” DNA and DNA condensedwith cationic lipids or polymers are typically delivered to the cellsusing chemical methods. A number of chemical methods are known in theart for cell delivery and include using lipids, polymers, or proteins tocomplex with DNA, optionally condensing the same into particles, anddelivering to the cells. Another non-viral chemical method includesusing cations to condense DNA, which is then placed in a liposome andused according to the present invention. See, C. Henry, 2001 Chemicaland Engineering News, 79(48):35-41.

[0078] The nucleic acid molecule encoding the CT-CRM of this inventionis introduced directly into the cells either as “naked” DNA (U.S. Pat.No. 5,580,859) or formulated in compositions with agents that facilitateimmunization, such as bupivicaine and other local anesthetics (U.S. Pat.No. 6,127,170).

[0079] All components of the viral and non-viral vectors above may bereadily selected from among known materials in the art and availablefrom the pharmaceutical industry. Selection of the vector components andregulatory sequences are not considered a limitation on this invention.Each nucleic acid sequence encoding a CT-CRM protein according to thisinvention is preferably under the control of regulatory sequences thatdirect the replication and generation of the product of each nucleicacid sequence in a mammalian or vertebrate cell. By the term“promoter/regulatory sequence” is meant a DNA sequence required forexpression of a nucleic acid operably linked thereto. Preferably thepromoter/regulatory sequence is positioned at the 5′ end of the codingsequence such that it drives expression of the CT-CRM protein in a cell.In some instances, the promoter/regulatory sequence may function in atissue specific manner. For example, the promoter/regulatory sequence isonly capable of driving expression in a cell of a particular tissuetype. In some instances, this sequence may be the core promoter sequenceand in other instances, this sequence may also include an enhancersequence and other regulatory elements that are required for expressionin a tissue-specific manner.

[0080] Suitable promoters may be readily selected from amongconstitutive promoters, inducible promoters, tissue-specific promotersand others. Examples of constitutive promoters that are non-specific inactivity and employed in the nucleic acid molecules encoding the CT-CRMprotein of this invention include, without limitation, the retroviralRous sarcoma virus (RSV) promoter, the retroviral LTR promoter(optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter(optionally with the CMV enhancer) (see, e.g., Boshart et al, Cell,41:521-530 (1985)), the SV40 promoter, the dihydrofolate reductasepromoter, the β-actin promoter, the phosphoglycerol kinase (PGK)promoter, and the EF1α promoter (Invitrogen).

[0081] Inducible promoters that are regulated by exogenously suppliedcompounds, include, without limitation, the arabinose promoter, thezinc-inducible sheep metallothionine (MT) promoter, the dexamethasone(Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7polymerase promoter system (WO 98/10088); the ecdysone insect promoter(No et al, 1996 Proc. Natl. Acad. Sci. USA, 93:3346-3351), thetetracycline-repressible system (Gossen et al, 1992 Proc. Natl. Acad.Sci. USA, 89:5547-5551), the tetracycline-inducible system (Gossen etal, 1995 Science, 268:1766-1769, see also Harvey et al, 1998 Curr. Opin.Chem Biol, 2:512-518), the RU486-inducible system (Wang et al, 1997 Nat.Biotech., 15:239-243 and Wang et al, 1997 Gene Ther., 4:432-441) and therapamycin-inducible system (Magari et al, 1997 J. Clin. Invest., 100:2865-2872). A particularly preferred promoter for use in expressionsystems for CT-CRMs is an arabinose inducible promoter.

[0082] Other types of inducible promoters that may be useful in thiscontext are those regulated by a specific physiological state, e.g.,temperature or acute phase or in replicating cells only. Usefultissue-specific promoters include the promoters from genes encodingskeletal β-actin, myosin light chain 2A, dystrophin, muscle creatinekinase, as well as synthetic muscle promoters with activities higherthan naturally-occurring promoters (see Li et al., 1999 Nat. Biotech.,17:241-245). Examples of promoters that are tissue-specific are knownfor liver (albumin, Miyatake et al. 1997 J. Virol., 71:5124-32;hepatitis B virus core promoter, Sandig et al., 1996 Gene Ther., 3:1002-9; alpha-fetoprotein (AFP), Arbuthnot et al., 1996 Hum. Gene Ther.,7:1503-14), bone (osteocalcin, Stein et al., 1997 Mol. Biol. Rep.,24:185-96; bone sialoprotein, Chen et al., 1996 J. Bone Miner. Res.,11:654-64), lymphocytes (CD2, Hansal et al., 1988 J. Immunol.,161:1063-8; immunoglobulin heavy chain; T cell receptor α chain),neuronal (neuron-specific enolase (NSE) promoter, Andersen et al. 1993Cell. Mol. Neurobiol., 13:503-15; neurofilament light-chain gene,Piccioli et al., 1991 Proc. Natl. Acad. Sci. USA, 88:5611-5; theneuron-specific ngf gene, Piccioli et al., 1995 Neuron, 15:373-84);among others. See, e.g., International Patent Publication No. WO00/55335for additional lists of known promoters useful, in this context.

[0083] Additional regulatory sequences for inclusion in a nucleic acidsequence, molecule or vector of this invention include, withoutlimitation, an enhancer sequence, a polyadenylation sequence, a splicedonor sequence and a splice acceptor sequence, a site for transcriptioninitiation and termination positioned at the beginning and end,respectively, of the polypeptide to be translated, a ribosome bindingsite for translation in the transcribed region, an epitope tag, anuclear localization sequence, an IRES element, a Goldberg-Hogness“TATA” element, a restriction enzyme cleavage site, a selectable markerand the like. Enhancer sequences include, e.g., the 72 bp tandem repeatof SV40 DNA or the retroviral long terminal repeats or LTRs, etc. andare employed to increase transcriptional efficiency. Selection ofpromoters and other common vector elements are conventional and manysuch sequences are available with which to design the nucleotidemolecules and vectors useful in this invention. See, e.g., Sambrook etal, Molecular Cloning. A Laboratory Manual, Cold Spring HarborLaboratory, New York, (1989) and references cited therein at, forexample, pages 3.18-3.26 and 16.17-16.27 and Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley & Sons, New York (1989). Oneof skill in the art may readily select from among such known regulatorysequences to prepare molecules of this invention. The selection of suchregulatory sequences is not a limitation of this invention.

[0084] C. Methods for Making the CT-CRM Proteins and NucleotideMolecules of this Invention

[0085] In view of the demonstrated utility of mutant CT-CRMs asadjuvants for antigenic compositions, production of suitable quantitiesof mutant CT-CRMs is desirable. The preparation or synthesis of thenucleotide sequences and CT-CRMs, as well as compositions containing thenucleotide molecules or CT-CRM protein of this invention disclosedherein is well within the ability of the person having ordinary skill inthe art using available material. The synthesis methods are not alimitation of this invention. The examples below detail presentlypreferred embodiments of synthesis of sequences encoding the CT-CRMs ofthis invention.

[0086] The CT-CRMs and nucleotide molecules and sequences of thisinvention may be produced by chemical synthesis methods, recombinantgenetic engineering methods, site directed mutagenesis, among others,and combinations of such methods. For example, the nucleotidesequences/CT-CRMs of the invention may be prepared conventionally byresort to known chemical synthesis techniques, e.g., solid-phasechemical synthesis, such as described by Merrifield, 1963 J. Amer. Chem.Soc., 85:2149-2154; J. Stuart and J. Young, Solid Phase PeptideSynthesis, Pierce Chemical Company, Rockford, Ill. (1984); Matteucci etal., 1981 J Am. Chem. Soc., 103:3185; Alvarado-Urbina et al., 1980Science, 214:270; and Sinha, N. D. et al., 1984 Nucl. Acids Res.,13:4539, among others. See, also, e.g., PROTEINS—STRUCTURE AND MOLECULARPROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, NewYork, 1993; Wold, F., “Posttranslational Protein Modifications:Perspectives and Prospects”, pgs. 1-12 in POSTTRANSLATIONAL COVALENTMODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York,1983; Seifter et al, 1990 Meth. Enzymol., 182:626-646, and Rattan et al,1992 Ann. N.Y. Acad. Sci., 663:48-62.

[0087] Alternatively, compositions of this invention may be constructedrecombinantly using conventional molecular biology techniques,site-directed mutagenesis, genetic engineering or polymerase chainreaction, such as, by cloning and expressing a nucleotide moleculeencoding a CT-CRM protein with optional other immunogens and optionalcarrier proteins within a host microorganism, etc. utilizing theinformation provided herein (See, e.g., Sambrook et al., cited above;Ausubel et al. cited above). Coding sequences for the CT-CRMs andoptional immunogens can be prepared synthetically (W. P. C. Stemmer etal, 1995 Gene, 164:49).

[0088] In general, recombinant DNA techniques involve obtaining bysynthesis or isolation a DNA sequence that encodes the CT-CRM protein asdescribed above, and introducing it into an appropriate vector/host cellexpression system where it is expressed preferably under the control ofan arabinose inducible promoter. Any of the methods described for theinsertion of DNA into an expression vector may be used to ligate apromoter and other regulatory control elements into specific siteswithin the selected recombinant vector. Suitable host cells are thentransformed, infected, transduced or transfected with such vectors orplasmids by conventional techniques.

[0089] A variety of host cell-vector (plasmid) systems may be used toexpress the immunogenic mutant cholera holotoxin. The vector system,which preferably includes the arabinose inducible promoter, iscompatible with the host cell used. The DNA encoding the mutant CT-CRMsare inserted into an expression system, and the promoter (preferably thearabinose inducible promoter), and other control elements are ligatedinto specific sites within the vector so that when the vector isinserted into a host cell (by transformation, transduction ortransfection, depending on the host cell-vector system used) the DNAencoding the CT-CRM is expressed by the host cell.

[0090] The vector may be selected from one of the viral vectors ornon-viral vectors described above but must be compatible with the hostcell used. The recombinant DNA vector may be introduced into appropriatehost cells (bacteria, virus, yeast, mammalian cells or the like) bytransformation, transduction or transfection (depending upon thevector/host cell system). Host-vector systems include but are notlimited to bacteria transformed with bacteriophage DNA, plasmid DNA orcosmid DNA; microorganisms such as yeast containing yeast vectors;mammalian cell systems infected with virus (e.g., vaccinia virus,adenovirus, etc.); and insect cell systems infected with virus (e.g.,baculovirus).

[0091] Systems for cloning and expressing the CT-CRMs and othercompositions of this invention using the synthetic nucleic acidmolecules include the use of various microorganisms and cells that arewell known in recombinant technology. The host cell may be selected fromany biological organism, including prokaryotic (e.g., bacterial) cellsand eukaryotic cells, including, mammalian, insect cells, yeast cells.Preferably, the cells employed in the various methods and compositionsof this invention are bacterial cells. Suitable bacterial cells include,for example, various strains of E. coli, Bacillus, and Streptomyces.Yeast cells such as Saccharomyces and Pichia, and insect cells such asSf9 and Sf21 cells are also useful host cells for production purposes.Mammalian cells including but not limited to Chinese hamster ovary cells(CHO), chick embryo fibroblasts, baby hamster kidney cells, NIH3T3, PERC6, NSO, VERO or COS cells are also suitable host cells, as well asother conventional and non-conventional organisms and plants.

[0092] The selection of other suitable host cells and methods fortransformation, culture, amplification, screening and product productionand purification can be performed by one of skill in the art byreference to known techniques. See, e.g., Gething and Sambrook, 1981Nature, 293:620-625, among others.

[0093] Typically, the host cell is maintained under culture conditionsfor a period of time sufficient for expression. Culture conditions arewell known in the art and include ionic composition and concentration,temperature, pH and the like. Typically, transfected cells aremaintained under culture conditions in a culture medium Suitable mediafor various cell types are well known in the art. In a preferredembodiment, temperature is from about 20° C. to about 50° C., morepreferably from about 30° C. to about 40° C. and, even more preferablyabout 37° C.

[0094] The pH is preferably from about a value of 6.0 to a value ofabout 8.0, more preferably from about a value of about 6.8 to a value ofabout 7.8 and, most preferably about 7.4. Osmolality is preferably fromabout 200 milliosmols per liter (mosm/L) to about 400 mosm/l and, morepreferably from about 290 mosm/L to about 310 mosm/L. Other biologicalconditions needed for transfection and expression of an encoded proteinare well known in the art.

[0095] Recombinant CT-CRM protein is recovered or collected either fromthe host cells or membranes thereof or from the medium in which thosecells are cultured. Recovery comprises isolating and purifying therecombinant CT-CRM protein. Isolation and purification techniques forpolypeptides are well known in the art and include such procedures asprecipitation, filtration, chromatography, electrophoresis and the like.

[0096] When produced by conventional recombinant means, CT-CRMs of thisinvention may be isolated and purified from the cell or medium thereofby conventional methods, including chromatography (e.g., ion exchange,affinity, and sizing column chromatography), centrifugation,differential solubility, or by any other standard techniques for thepurification or proteins. Several techniques exist for purification ofheterologous protein from prokaryotic cells. See, U.S. Pat. Nos.4,518,526; 4,599,197; and 4,734,362. The purified preparation howeverproduced should be substantially free of host toxins, which might beharmful to humans. In particular, when expressed in gram negativebacterial host cells such as E coli, the purified peptide or proteinshould be substantially free of endotoxin contamination. See, e.g.,Sambrook et al., cited above.

[0097] The CT-CRMs used in methods and compositions of the invention arenot limited to products of any of the specific exemplary processeslisted herein. In fact, the protein may be prepared by the methods inthe texts cited immediately above or by methods of the texts citedelsewhere in this specification It is within the skill of the art toisolate and produce recombinantly or synthetically protein compositionsfor such use.

[0098] The five exemplary CT-CRMs of Table 1, two bearing a single aminoacid substitution, one bearing a single amino acid insertion, and twobearing double amino acid substitutions were generated as described indetail in Example 1 using some of the methods described above.Specifically, a set of mutant CT clones (CT-CRMs) were generated in E.coli by standard site-directed mutagenesis protocols on plasmidsencoding the known CT holotoxin molecules. It has previously been shownthat the resulting yield of purified CT-CRM_(E29H) holotoxin wasapproximately 50 μg per liter of culture medium (see Internationalpatent publication No. WO 00/18434). Initial attempts to increaseCT-CRM_(E29H) yield via modifications to the original plasmid, showedlittle or no effect. A moderate increase in yield was achieved throughco-expression of the plasmid pIIB29H, and derivatives, with Vibriocholerae DsbA and E. coli RpoH. Co-expression and purificationmodifications increased the yield of CT-CRM_(E29H) to approximately 2mg/liter.

[0099] In order to increase the expression of CT-CRMs of the presentinvention, the lactose inducible promoter in the plasmids was replacedwith an arabinose inducible promoter (Invitrogen Corporation, Carlsbad,Calif.), which was operatively linked to the DNA sequence encoding theCT-CRMs. During cloning it was determined that plasmid pIIB29H containeda ctxA gene encoding CT subunit A from Vibrio cholerae strain 569B,linked to a ctxB gene encoding CT subunit B from Vibrio cholerae strain2125. Cross alignment of these genes indicated seven base substitutionsbetween the two ctxB genes and a single base change between the ctxAgenes. Several of these base substitutions led to amino acid changes inthe mature subunits. Of special note is the substitution between thectxA genes which leads to an amino acid change within the A-2 portion,or the holotoxin assembly domain of the A subunit. It was not knownwhether the heterogeneity between these genes had a negative impact ontoxin expression or holotoxin assembly. However, it was thoughtpreferable from an evolutionary standpoint that both toxin subunit genesoriginate from the same source. As such, both the ctxA and ctxB genesused in the construction of the arabinose inducible system originatedfrom Vibrio cholerae strain 569B. The construction of plasmids pLP911,pLP915, pLP907, pLP909 and pLP910 is described in Example 1. Theimmunogenic mutant cholera holotoxin is produced by transforming,infecting, transducing or transfecting a host cell with a plasmiddescribed above, and culturing the host cell under conditions thatpermit the expression of said recombinant immunogenic detoxified proteinby the host cell. The yield of CT-CRMs from pLP911, pLP915, pLP907,pLP909 and pLP910 is approximately 7.6, 5.6, 7.9, 27.4, and 1.9 mg ofpurified material per liter of culture, respectively.

[0100] The resulting CT-CRM protein or nucleic acid molecule may beformulated into an immunogenic composition with any number of selectedantigens and screened for adjuvant efficacy by in vivo assays, such asthose described in the examples below.

[0101] D. Immunogenic Compositions

[0102] An effective immunogenic composition according to the inventionis one comprising a mutant cholera holotoxin of this invention.Preferably the mutant cholera holotoxin CT-CRM has reduced toxicitycompared to a wild-type cholera holotoxin. This “reduced toxicity”enables each mutant to be used as an adjuvant in an immunogeniccomposition without causing significant side effects, particularly thoseknown to be associated with wild-type CT, e.g., diarrhea. Morepreferably, the CT-CRM in the immunogenic composition of this inventionhas a single amino acid substitution (arginine to tryptophan or arginineto glycine) at amino acid position 25 in the A subunit (CT-CRM_(R25W),CT-CRM_(R25G)). In another preferred embodiment, the CT-CRM has a singleamino acid insertion of histidine in the amino acid position 49 adjacentto the amino acid residue threonine at the amino acid position 48 in theA subunit (CT-CRM_(T48TH)). A third preferred embodiment is a CT-CRMwith a double amino acid insertion of amino acid residues glycine andproline in the amino acid positions 35 and 36 adjacent to the amino acidresidue glycine at the amino acid position 34 in the A subunit(CT-CRM_(G34GGP)). A fourth exemplary CT-CRM has a single amino acidsubstitution at the amino acid position 30 (tyrosine for tryptophan) anda double amino acid insertion of amino acid residues alanine andhistidine in the amino acid positions 31 and 32 adjacent to the aminoacid residue at the amino acid position 30 in the A subunit(CT-CRM_(Y30WAH)). In one embodiment, the CT-CRM may have one or moreadditional modifications as described above. In another embodiment, thecomposition comprises a selected antigen and a suitable effectiveadjuvanting amount of the CT-CRM, wherein said holotoxin significantlyenhances the immune response in a vertebrate host to said antigen. Thecompositions of the present invention modulate the immune response byimproving the vertebrate host's antibody response and cell-mediatedimmune responses to the administration of a composition comprising aselected antigen as described above.

[0103] As used herein, the term “effective adjuvanting amount” means adose of one of the CT-CRM mutants of this invention that is effective ineliciting an increased immune response in a vertebrate host. In a morespecific definition, the term “effective adjuvanting amount” means adose of one of the five CT-CRM mutants described herein (CT-CRM_(R25W,)CT-CRM_(R25G), CT-CRM_(T48TH), CT-CRM_(G34GGP), CT-CRM_(Y30WAH)),effective in eliciting an increased immune response in a vertebratehost. Specifically, the CT-CRMs disclosed herein augment mucosal andsystemic immune responses following intranasal administration ofdisparate antigens. Furthermore, even in the presence of pre-existinganti-CT immune responses, the mutant CT-CRMs were able to serve asefficient mucosal adjuvants. The immunogenic mutant CT-CRMs according tothe present invention exhibit a balance of reduced toxicity and retainedadjuvanticity, such that the resulting mutant CT protein functions as anadjuvant while being tolerated safely by the vertebrate host to which itis introduced. The particular “effective adjuvanting dosage or amount”will depend upon the age, weight and medical condition of the host, aswell as on the method of administration Suitable doses are readilydetermined by persons skilled in the art.

[0104] The immunogenic compositions containing as an adjuvant the mutantcholera holotoxins of this invention also contain at least one antigenselected from among a wide variety of antigens. The antigen(s) maycomprise a whole cell or virus, or one or more saccharides, proteins,protein subunits, polypeptide, peptide or fragments, poly- oroligonucleotides, or other macromolecular components. If desired, theantigenic compositions may contain more than one antigen from the sameor different pathogenic microorganisms.

[0105] Thus, in one embodiment, the immunogenic compositions of thisinvention comprise as the selected antigen a polypeptide, peptide orfragment derived from a pathogenic bacterium. Desirable bacterialimmunogenic compositions including the CT-CRM mutant(s) as an adjuvantinclude those directed to the prevention and/or treatment of disease(s)caused by, without limitation, Haemophilus influenzae (both typable andnontypable), Haemophilus somnus, Moraxella catarralis, Streptococcuspneumoniae, Streptococcus pyogenes, Streptococcus agalactiae,Streptococcus faecalis, Helicobacter pylori, Neisseria meningitidis,Neisseria gonorrhoeae, Chlamydia trachomatis, Chlamydia pneumoniae,Chlamydia psittaci, Bordetella pertussis, Alloiococcus otiditis,Salmonella typhi, Salmonella typhimurium, Salmonella choleraesuis,Escherichia coli, Shigella, Vibrio cholerae, Corynebacteriumdiphtheriae, Mycobacterium tuberculosis, Mycobacteriumavium-Mycobacterium intracellulare complex, Proteus mirabilis, Proteusvulgaris, Staphylococcus aureus, Staphylococcus epidermidis, Clostridiumtetani, Leptospira interrogans, Borrelia burgdorferi, Pasteurellahaemolytica, Pasteurella multocida, Actinobacillus pleuropneumoniae andMycoplasma gallisepticum.

[0106] In another embodiment, the immunogenic compositions of thisinvention comprise as the selected antigen a polypeptide, peptide orfragment derived from a pathogenic virus. Desirable viral immunogeniccompositions including the CT-CRM mutant(s) as an adjuvant include thosedirected to the prevention and/or treatment of disease caused by,without limitation, Respiratory syncytial virus, Parainfluenza virustypes 1-3, Human metapneumovirus, Influenza virus, Herpes simplex virus,Human cytomegalovirus, Human immunodeficiency virus, Simianimmunodeficiency virus, Hepatitis A v Hepatitis B virus, Hepatitis Cvirus, Human papillomavirus, Poliovirus, rotavirus, caliciviruses,Measles virus, Mumps virus, Rubella virus, adenovirus, rabies virus,canine distemper virus, rinderpest virus, avian pneumovirus (formerlyturkey rhinotracheitis virus), Hendra virus, Nipah virus, coronavirus,parvovirus, infectious rhinotracheitis viruses, feline leukemia virus,feline infectious peritonitis virus, avian infectious bursal diseasevirus, Newcastle disease virus, Marek's disease virus, porcinerespiratory and reproductive syndrome virus, equine arteritis virus andvarious Encephalitis viruses.

[0107] In another embodiment, the immunogenic compositions of thisinvention comprise as the selected antigen a polypeptide, peptide orfragment derived from a pathogenic fungus. Desirable immunogeniccompositions against fungal pathogens including the CT-CRM mutant(s) asan adjuvant include those directed to the prevention and/or treatment ofdisease(s) caused by, without limitation, Aspergillis, Blastomyces,Candida, Coccidiodes, Cryptococcus and Histoplasma.

[0108] In still another embodiment, the immunogenic compositions of thisinvention comprise as the selected antigen a polypeptide, peptide orfragment derived from a pathogenic parasite. Desirable immunogeniccompositions against parasites including the CT-CRM mutant(s) as anadjuvant include those directed to the prevention and/or treatment ofdisease(s) caused by, without limitation, Leishmania major, Ascaris,Trichuris, Giardia, Schistosoma, Cryptosporidium, Trichomonas,Toxoplasma gondii and Pneumocystis carinii.

[0109] Desirable immunogenic compositions directed againstnon-infectious diseases including the CT-CRM mutant(s) as an adjuvantare also within the scope of this invention. Such immunogeniccompositions include those directed to vertebrate antigens, particularlycompositions directed against antigens for the prevention and/ortreatment of disease(s), without limitation, such as allergy, autoimmunedisease, Alzheimer disease and cancer.

[0110] For example, the immunogenic composition of this invention maycontain a polypeptide, peptide or fragment derived from a cancer cell ortumor cell. Desirable immunogenic compositions for eliciting atherapeutic or prophylactic anti-cancer effect in a vertebrate host,which contain the CT-CRM mutants of this invention, include thoseutilizing a cancer antigen or tumor-associated antigen including,without limitation, prostate specific antigen, carcino-embryonicantigen, MUC-1, Her2, CA-125, MAGE-3, hormones, hormone analogs and soforth.

[0111] Other immunogenic compositions of this invention are desirablefor moderating responses to allergens in a vertebrate host. Suchcompositions contain the CT-CRM mutant(s) of this invention and apolypeptide, peptide or fragment derived from an allergen or fragmentthereof. Examples of such allergens are described in the U.S. Pat. No.5,830,877 and International patent publication No. WO 99/51259, whichare hereby incorporated by reference, and include pollen, insect venoms,animal dander, fungal spores and drugs (such as penicillin). Theimmunogenic compositions interfere with the production of IgEantibodies, a known cause of allergic reactions, so as to moderateallergic responses to the allergen.

[0112] In still another embodiment, the immunogenic compositions of thisinvention contain as the selected antigen a polypeptide, peptide orfragment derived from a molecular portion of an antigen, whichrepresents those produced by a host (a self molecule) in an undesiredmanner, amount or location, such as those from amyloid precursor proteinso as to prevent or treat disease characterized by amyloid deposition ina vertebrate host. Desirable compositions for moderating responses toself molecules in a vertebrate host, which contain CT-CRM mutants ofthis invention, include those containing a self molecule or fragmentthereof. Examples of such self molecules include β-chain insulininvolved in diabetes, the G17 molecule involved in gastroesophagealreflux disease, and antigens which downregulate autoimmune responses indiseases such as multiple sclerosis, lupus and rheumatoid arthritis.

[0113] Still other immunogenic compositions of this invention aredesirable for preventing or treating disease characterized by amyloiddeposition in a vertebrate host. Such compositions contain the CT-CRMmutant(s) of this invention as well as portions of amyloid precursorprotein (APP). This disease is referred to variously as Alzheimer'sdisease, amyloidosis or amyloidogenic disease. The β-amyloid precursorprotein (also referred to as Aβ peptide) is a 42 amino acid fragment ofAPP, which is generated by processing of APP by the β and γ secretaseenzymes, and has the following sequence: Asp Ala Glu Phe Arg His Asp SerGly Tyr Glu Val His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly SerAsn Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala (SEQ IDNO: 3). In some patients, the amyloid deposit takes the form of anaggregated Aβ peptide. Surprisingly, it has now been found thatadministration of isolated Aβ peptide induces an immune response againstthe Aβ peptide component of an amyloid deposit in a vertebrate host(International patent publication No. WO 99/27944). Thus, embodiments ofthis invention include the CT-CRM mutants of this invention plus Aβpeptide, as well as fragments of Aβ peptide and antibodies to Aβpeptides or fragments thereof. One such fragment of Aβ peptide is the 28amino acid peptide having the following sequence (U.S. Pat. No.4,666,829): Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His GlnLys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys (SEQ ID NO: 4).

[0114] Such immunogenic compositions further comprise an immunologicallyacceptable diluent or a pharmaceutically acceptable carrier, such assterile water or sterile isotonic saline. The antigenic compositions mayalso be mixed with such diluents or carriers in a conventional manner.As used herein the language “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with administration to humans or othervertebrate hosts. The appropriate carrier will be evident to thoseskilled in the art and will depend in large part upon the route ofadministration.

[0115] The immunogenic compositions may also include, but are notlimited to, suspensions, solutions, emulsions in oily or aqueousvehicles, pastes, and implantable sustained-release or biodegradableformulations. Such formulations may further comprise one or moreadditional ingredients including, but not limited to, suspending,stabilizing, or dispersing agents. In one embodiment of a formulationfor parenteral administration, the active ingredient is provided in dry(i e., powder or granular) form for reconstitution with a suitablevehicle (e.g., sterile pyrogen-free water) prior to parenteraladministration of the reconstituted composition. Otherparenterally-administrable formulations, which are useful, includethose, which comprise the active ingredient in microcrystalline form, ina liposomal preparation, or as a component of a biodegradable polymersystem. Compositions for sustained release or implantation may comprisepharmaceutically acceptable polymeric or hydrophobic materials such asan emulsion, an ion exchange resin, a sparingly soluble polymer, or asparingly soluble salt.

[0116] Still additional components that may be present in the proteinimmunogenic compositions of this invention are adjuvants in addition tothe CT-CRMs, preservatives, chemical stabilizers, or other antigenicproteins. Typically, stabilizers, adjuvants, and preservatives areoptimized to determine the best formulation for efficacy in the targethuman or animal. Suitable exemplary preservatives include chlorobutanol,potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, theparabens, ethyl vanillin, glycerin, phenol, and parachlorophenol.Suitable stabilizing ingredients that may be used include, for example,casamino acids, sucrose, gelatin, phenol red, N-Z amine, monopotassiumdiphosphate, lactose, lactalbumin hydrolysate, and dried milk.

[0117] The antigenic compositions of this invention may comprise furtheradjuvants in addition to the mutant CT-CRMs. Conventional non-CT-CRMadjuvants used to enhance an immune response include, withoutlimitation, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa,Hamilton, Mont.), which is described in U.S. Pat. No. 4,912,094, whichis hereby incorporated by reference. Also suitable for use as adjuvantsare synthetic lipid A analogs or aminoalkyl glucosamine phosphatecompounds (AGP), or derivatives or analogs thereof, which are availablefrom Corixa (Hamilton, Mont.), and which are described in U.S. Pat. No.6,113,918, which is hereby incorporated by reference. One such AGP is2-[(R)-3-Tetradecanoyloxytetradecanoylamino]ethyl2-Deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyoxytetradecanoyl]-2-[(R)-3-tetradecanoyoxytetradecanoyl-amino]-b-D-glucopyranoside,which is also known as 529 (formerly known as RC529). This 529 adjuvantis formulated as an aqueous form or as a stable emulsion.

[0118] Still other non-CT-CRM adjuvants include mineral oil and wateremulsions, aluminum salts (alum), such as aluminum hydroxide, aluminumphosphate, etc., Amphigen, Avridine, L121/squalene,D-lactide-polylactide/glycoside, pluronic polyols, muramyl dipeptide,killed Bordetella, saponins, such as Stimulon™ QS-21 (Antigenics,Framingham, Mass.), described in U.S. Pat. No. 5,057,540, which ishereby incorporated by reference, and particles generated therefrom suchas ISCOMS (immunostimulating complexes), Mycobacterium tuberculosis,bacterial lipopolysaccharides, synthetic polynucleotides such asoligonucleotides containing a CpG motif (U.S. Pat. No. 6,207,646, whichis hereby incorporated by reference), a pertussis toxin (PT), or an E.coli heat-labile toxin (LT), particularly LT-K63, LT-R72, CT-S109,PT-K9/G129; see, e.g., International Patent Publication Nos. WO 93/13302and WO 92/19265, incorporated herein by reference.

[0119] Various cytokines and lymphokines are also suitable for inclusionin the immunogenic compositions of this invention. One such cytokine isgranulocyte-macrophage colony stimulating factor (GM-CSF), which has anucleotide sequence as described in U.S. Pat. No. 5,078,996, which ishereby incorporated by reference. A plasmid containing GM-CSF cDNA hasbeen transformed into E. coli and has been deposited with the AmericanType Culture Collection (ATCC), 10801 University Boulevard, Manassas,Va. 20110-2209, under Accession Number 39900. The cytokineInterleukin-12 (IL-12) is another adjuvant that is described in U.S.Pat. No. 5,723,127, which is hereby incorporated by reference (availablefrom Genetics Institute, Inc., Cambridge, Mass.). Other cytokines orlymphokines have been shown to have immune modulating activity,including, but not limited to, the interleukins 1-α, 1-β, 2, 4, 5, 6, 7,8, 10, 13, 14, 15, 16, 17 and 18, the interferons-α, β and γ,granulocyte colony stimulating factor, and the tumor necrosis factors αand β, and are suitable for use as adjuvants.

[0120] Still other suitable optional components of the immunogeniccompositions of this invention include, but are not limited to: surfaceactive substances (e.g., hexadecylamine, octadecylamine, octadecyl aminoacid esters, lysolecithin, dimethyl-dioctadecylammonium bromide),methoxyhexadecylgylcerol, and pluronic polyols; polyamines, e.g., pyran,dextransulfate, poly IC, carbopol; peptides, e.g., muramyl dipeptide,dimethylglycine, tuftsin; oil emulsions; and mineral gels, e.g.,aluminum phosphate, etc. and immune stimulating complexes. The CT-CRMand antigen may also be incorporated into liposomes, or conjugated topolysaccharides, lipopolysaccharides and/or other polymers for use in animmunogenic composition.

[0121] Immunogenic compositions of this invention including the CT-CRMmutant(s), or DNA sequences and molecules encoding the desired CT-CRM ofthis invention, are also useful as polynucleotide compositions (alsoknown as DNA immunogenic compositions) or administered withpolynucleotides encoding the selected antigen. For example, it has beenpreviously demonstrated that BALB/c mice administered a formulation ofplasmid DNA (pDNA) encoding the full length glycoprotein D of herpessimplex virus (HSV) type 2 (gD2), along with CT-CRM_(E29H) by theintradermal route generated a higher average cellular response thanthose that received plasmid DNA encoding HSV gD2 by itself by theintradermal route. In addition, the average serum antibody titers formice, which, received the plasmid DNA HSV gD2 composition along withCT-CRM_(E29H) was approximately the same as that seen in mice thatreceived the plasmid DNA HSV gD2 composition without adjuvant.Similarly, the plasmid DNA HSV gD2 composition adjuvanted withCT-CRM_(E29H) also generated a gD2-specific antibody response in vaginalwash samples at levels that were comparable to those seen following thedelivery of the non-adjuvanted composition by intradermal orintramuscular routes. Mice immunized with the plasmid DNA HSV gD2composition adjuvanted with CT-CRM_(E29H) or CT and delivered by theintradermal route also generated substantially higher levels of gammainterferon and IL-5 than mice that received the plasmid DNA HSV-gD2composition without adjuvant. Thus, CT-CRMs enhance proliferative andgamma interferon responses when administered with a plasmid DNAcomposition against HSV.

[0122] In addition to a carrier as described above, immunogeniccompositions composed of polynucleotide molecules desirably containoptional polynucleotide facilitating agents or “co-agents”, such as alocal anesthetic, a peptide, a lipid including cationic lipids, aliposome or lipidic particle, a polycation such as polylysine, abranched, three-dimensional polycation such as a dendrimer, acarbohydrate, a cationic amphiphile, a detergent, a benzylammoniumsurfactant, or another compound that facilitates polynucleotide transferto cells. Such a facilitating agent includes bupivicaine (see U.S. Pat.No. 5,593,972, which is hereby incorporated by reference). Othernon-exclusive examples of such facilitating agents or co-agents usefulin this invention are described in U.S. Pat. Nos. 5,703,055; 5,739,118;5,837,533; International Patent Publication No. WO96/10038, publishedApr. 4, 1996; and International Patent Publication No WO94/16737,published Aug. 8, 1994, which are each incorporated herein by reference.

[0123] Most preferably, the local anesthetic is present in an amountthat forms one or more complexes with the nucleic acid molecules. Whenthe local anesthetic is mixed with nucleic acid molecules or plasmids ofthis invention, it forms a variety of small complexes or particles thatpack the DNA and are homogeneous. Thus, in one embodiment of theimmunogenic compositions of this invention, the complexes are formed bymixing the local anesthetic and at least one plasmid of this invention.Any single complex resulting from this mixture may contain a variety ofcombinations of the different plasmids. Alternatively, in anotherembodiment of the compositions of this invention, the local anestheticmay be pre-mixed with each plasmid separately, and then the separatemixtures combined in a single composition to ensure the desired ratio ofthe plasmids is present in a single immunogenic composition, if allplasmids are to be administered in a single bolus administration.Alternatively, the local anesthetic and each plasmid may be mixedseparately and administered separately to obtain the desired ratio.Where, hereafter, the term “complex” or “one or more complexes” or“complexes” is used to define this embodiment of the immunogeniccomposition, it is understood that the term encompasses one or morecomplexes with each complex containing a mixture of the CT-CRM-encodingplasmids and antigen-encoding plasmids, or a mixture of complexes formeddiscretely, wherein each complex contains only one type of plasmid, or aone or a mixture of complexes wherein each complex contains apolycistronic DNA Preferably, the complexes are between about 50 toabout 150 nm in diameter. When the facilitating agent used is a localanesthetic, preferably bupivacaine, an amount of from about 0.1 weightpercent to about 1.0 weight percent based on the total weight of thepolynucleotide composition is preferred. See, also, International PatentPublication No. WO99/21591, which is hereby incorporated by reference,and which teaches the incorporation of benzylammonium surfactants asco-agents, preferably administered in an amount of between about0.001-0.03 weight %. According to the present invention, the amount oflocal anesthetic is present in a ratio to said nucleic acid molecules of0.01-2.5% w/v local anesthetic to 1-10 μg/ml nucleic acid. Another suchrange is 0.05-1.25% w/v local anesthetic to 100 μg/ml to 1 ml/ml nucleicacid.

[0124] As used, such a polynucleotide immunogenic composition expressesthe CT-CRM and antigens on a transient basis in vivo; no geneticmaterial is inserted or integrated into the chromosomes of the host.This use is thus distinguished from gene therapy, where the goal is toinsert or integrate the genetic material of interest into thechromosome. An assay is used to confirm that the polynucleotidesadministered by immunization do not rise to a transformed phenotype inthe host (U.S. Pat. No. 6,168,918).

[0125] The immunogenic compositions may also contain other additivessuitable for the selected mode of administration of the composition. Thecomposition of the invention may also involve lyophilizedpolynucleotides, which can be used with other pharmaceuticallyacceptable excipients for developing powder, liquid or suspension dosageforms. See, e.g., Remington: The Science and Practice of Pharmacy, Vol.2, 19^(th) edition (1995), e.g., Chapter 95 Aerosols; and InternationalPatent Publication No. WO99/45966, the teachings of which are herebyincorporated by reference. Routes of administration for thesecompositions may be combined, if desired, or adjusted.

[0126] These nucleic acid molecule-containing immunogenic compositionscan contain additives suitable for administration via any conventionalroute of administration. In some preferred embodiments, the immunogeniccomposition of the invention is prepared for administration to humansubjects in the form of, for example, liquids, powders, aerosols,tablets, capsules, enteric-coated tablets or capsules, or suppositories.

[0127] The immunogenic compositions of the present invention (whetherprotein-containing or nucleic acid molecule-containing compositions), asdescribed above, are not limited by the selection of the conventional,physiologically acceptable, carriers, adjuvants, or other ingredientsuseful in pharmaceutical preparations of the types described above. Thepreparation of these pharmaceutically acceptable compositions, from theabove-described components, having appropriate pH isotonicity, stabilityand other conventional characteristics is within the skill of the art.

[0128] E. Methods of Use of the Compositions of this Invention

[0129] The immunogenic compositions of this invention that comprise theCT-CRM alone or a combination of the CT-CRM and a selected antigen, areadministered to a human or to a non-human vertebrate by a variety ofroutes to enhance the immune response to an antigen, preferably adisease-causing antigen, as identified above. The compositions of thepresent invention modulate the immune response by improving thevertebrate host's antibody response and cell-mediated immunity afteradministration of a composition comprising a selected antigen asdescribed above, and an effective adjuvanting amount of a mutant CT-CRM,where the mutant CT-CRM has substantially reduced toxicity compared to awild-type CT, and wherein the reduced toxicity is a result of a singleamino acid substitution, a single amino acid insertion, a double aminoacid insertion or a single amino acid substitution and double amino acidinsertion.

[0130] In one embodiment, the immunogenic composition containing theCT-CRM (either as a protein or encoded by a nucleic acid molecule) isadministered prior to administration of a composition comprising theselected antigen (either as a protein or as a nucleic acid). In anotherembodiment, the immunogenic composition is administered simultaneouslywith the antigen, whether it is administered in a composition containingboth antigen and CT-CRM or as a separate composition from that of theantigen-containing composition. In still a further embodiment, thecomposition containing the CT-CRM is administered after the compositioncontaining the antigen. It is preferable, although not required, thatthe antigen and the mutant CT-CRM be administered at the same time.

[0131] The immunogenic composition containing the CT-CRM may beadministered as a protein or as a nucleic acid molecule encoding theprotein, as described above. The immunogenic composition containing theCT-CRM may be administered as a protein in combination with a selectedantigen administered as a protein. Alternatively, as described above,the CT-CRM immunogenic composition may be administered as a protein witha nucleic acid molecule encoding the antigen, as described above. Stillanother alternative involves administering both the CT-CRM and theantigen as nucleic acid sequences encoding these proteins.

[0132] Any suitable route of administration may be employed toadminister the immunogenic composition containing the CT-CRM. The routemay be the same or different from a route selected to administer acomposition containing the selected antigen, if the CT-CRM and antigenare administered in separate compositions or in different forms, e.g.,protein or nucleic acids. Suitable routes of administration include, butare not limited to, intranasal, oral, vaginal, rectal, parenteral,intradermal, transdermal (see, e.g., International patent publicationNo. WO 98/20734, which is hereby incorporated by reference),intramuscular, intraperitoneal, subcutaneous, intravenous andintraarterial. The appropriate route is selected depending on the natureof the immunogenic composition used, and an evaluation of the age,weight, sex and general health of the patient and the antigens presentin the immunogenic composition, and similar factors by an attendingphysician.

[0133] In general, selection of the appropriate “effective amount” ordosage for the the CT-CRM and/or antigen components of the immunogeniccomposition(s) of the present invention will also be based upon theprotein or nucleic acid form of the CT-CRM and antigen, the identity ofthe antigen in the immunogenic composition(s) employed, as well as thephysical condition of the subject, most especially including the generalhealth, age and weight of the immunized subject. The method and routesof administration and the presence of additional components in theimmunogenic compositions may also affect the dosages and amounts of theCT-CRM and antigen. Such selection and upward or downward adjustment ofthe effective dose is within the skill of the art. The amount of CT-CRMand antigen required to induce an immune response, preferably aprotective response, or produce an exogenous effect in the patientwithout significant adverse side effects varies depending upon thesefactors. Suitable doses are readily determined by persons skilled in theart.

[0134] As an example, in one embodiment, for the compositions containingprotein components, e.g., a CT-CRM variant protein and/or antigen asdescribed above, each dose may comprise between about 1 μg to about 20mg of the protein per mL of a sterile solution. Other dosage ranges mayalso be contemplated by one of skill in the art. Initial doses may beoptionally followed by repeated boosts, where desirable.

[0135] In another example, the amounts of nucleotide molecules in theDNA and vector compositions may be selected and adjusted by one of skillin the art. In one embodiment, each dose will comprise between about 50μg to about 1 mg of CT-CRM-encoding or antigen-encoding nucleic acid,e.g., DNA plasmid, per mL of a sterile solution.

[0136] The number of doses and the dosage regimen for the compositionare also readily determined by persons skilled in the art. Protectionmay be conferred by a single dose of the immunogenic compositioncontaining the CT-CRM, or may require the administration of severaldoses with or without the selected antigen, in addition to booster dosesat later times to maintain protection. In some instances, the adjuvantproperty of the mutant CT-CRM may reduce the number of doses containingantigen that are needed or may reduce the time course of the dosageregimen. The levels of immunity can be monitored to determine the need,if any, for boosters.

[0137] In order that this invention may be better understood, thefollowing examples are set forth. The examples are for the purpose ofillustration only and are not to be construed as limiting the scope ofthe invention.

[0138] All references cited herein are hereby incorporated by reference.

EXAMPLE 1 Expression of CT Mutants

[0139] A. Bacterial Strains, Plasmids and Growth Conditions.

[0140]E. coli TG1 (Amersham Corporation, Arlington Heights, Ill.), TX1,a naladixic-acid resistant derivative of TG1 carrying F′Tc, lacI^(q)from XL1blue (Stratagene, La Jolla, Calif.), TE1 (TG1 ends, F′Tc,lacI^(q)) and CJ236(F′Tc, lacI^(q)) (BioRad, Hercules, Calif.) were usedas hosts for cloning recombinant plasmids and expression of variantproteins. Plasmid-containing strains were maintained on LB agar plateswith antibiotics as required (ampicillin, 50 μg/ml; kanamycin 25 μg/ml;tetracycline 10 μg/ml).

[0141] B. Mutagenesis of ctxA Gene.

[0142] Site-directed mutagenesis using single-stranded uracil-containingtemplates (Jobling, M. G. and Holmes, R. K., 1992 Infect. Immun.,60:4915-24) was used to select for oligonucleotide-derived mutantscreated in plasmid pMGJ67, a clone of the native CT operon in pSKII-(Stratagene). Briefly, each oligonucleotide was phosphorylated and usedto direct second strand synthesis on a single-stranded DNA templaterescued from dut ung CJ236 (F′Tc, pMGJ67). Following ligation andtransformation of ung⁺ strain TX1, single-stranded DNA was rescued fromAp^(R) transformants and sequenced by the dideoxy chain terminationmethod (Kunkel, T. A., 1985 Proc. Natl. Acad., Sci., USA, 82:488-492).Some mutations were introduced directly into pARCT2 using theQuickChange mutagenesis method (Stratagene). pARCT2 is anarabinose-inducible clone derived from pAR3 (International PatentPublication No. WO98/20734) expressing an operon containing the ctxA andctxB genes with signal sequences derived from the LTIIb B gene, and witheach gene independently using the translation inititation sequencesderived from T7 gene 10 from vector plasmid pT7-7, a derivative ofpT7-1.

[0143] C. One and Two Codon Insertion Mutations.

[0144] Single codon insertions were generated at DdeI restriction sitesby partial digestion of pMGJ64 (a derivative of pMGJ67), followed byfilling-in of the 3-base sticky ends and self-ligation. Two codonTAB-linker insertion mutations were made by adding six base-pair ApaIlinkers (GGGCCC) to the ends of RsaI partial digests of pMGJ64 asdescribed in the TAB manual (Pharmacia). Transformants were screened forloss of either a single DdeI or RsaI site (and presence of a new ApaIsite) and confirmed by DNA sequencing.

[0145] D. Construction of Arabinose Promoted CT-CRM Expression Vectors.

[0146] Previous experience with CT-CRM_(E29H) (International PatentPublication No. WO 00/18434) has shown that maximal production in E.coli could be achieved by substituting synthetic Shine-Delgaro sequencesupstream of the ctxA gene and placing the operon under the control ofthe arabinose promoter system. CT operons containing site directedmutations in the A subunit were made as previously described (supra).CT-CRMs were originally under the control of a 3-galactosidase promoterand expression levels in E. coli were low. PCR was used to modify theregion 5′ to the ATG of the CT-A subunit and insert an NheI site at the5′ end. The corresponding 3′ primer added a HindIII site at the 3′ endof the CT-B gene. Primer sequences used were: CT29FNhe:5′ TTTTTTGGGCTAGCATGGAGGAAAAGATGAGC (SEQ ID NO: 5) CT29RHnd:5′ CGAGGTCGAAGCTTGCATGTTTGGGC. (SEQ ID NO: 6)

[0147] PCR was performed on each mutant CT-CRM operon and the PCRproducts were ligated into pCR2.1-Topo (Invitrogen) according to themanufacturer's directions and transformed into Top10F′ cells.Recombinant E. coli were plated onto SOB agar containing Kanamycin (25μg/ml) and X-gal (40 μg/ml). Plasmids from white colonies were screenedfor inserts by digestion with EcoRI. Plasmids containing inserts of thecorrect size were digested with NheI and HindIII according to themanufacturer's directions and the DNA fragments containing the CToperons isolated from low melting point agarose. Plasmid pBAD 18-Cm(Invitrogen) was digested with NheI-HindIII and the linear DNA isolatedfrom low melting point agarose. Digested pBAD18 and the CT operons wereligated at 12° C. and transformed into Top10F E coli. Plasmids fromchloramphenicol-resistant colonies were screened for inserts byrestriction analysis, and representative clones were sequenced toconfirm the presence of the site directed mutations. Plasmids weretransformed into DH5α for expression of CT-CRMs.

[0148] E. Expression of CT-CRMs in E. coli.

[0149]E. coli DH5α cells containing plasmids pLP9911, pLP915, pLP907,pLP909 and pLP910, cells expressing the CT-CRMs respectively, were grownm phosphate buffered Hy-Soy media containing chloramphenicol (25 μg/ml)and glycerol (0.5%) at 37° C. with aeration. When cultures reached anOD₆₀₀ of approximately 4.5-5.5, they were induced by addition ofL-arabinose to a final concentration of 0.5%. Cultures were incubated at37° C. with aeration for three hours post-induction and then the cellscollected by centrifugation. Cell pellets were stored at −20° C.

[0150] F Preparation and Purification of CT-CRMs.

[0151] Cell pellets were thawed at room temperature and resuspended in10 mM NaPO₄ and 1 mM EDTA (pH 7.0) at 9% of the original culture volume.Cell suspensions were mechanically disrupted in a microfluidizer andcentrifuged for 10 minutes at 8,500×g. Cell lysates were furtherclarified at 160,000×g for one hour. The clarified cell lysate wasloaded, at a flow rate of 2 ml/minute, onto a carboxymethyl(CM)-sepharose5 column (300 ml CM-Sepharose™ per 10 liters of culture)(Amersham, Pharmacia) equilibrated with 10 mM NaPO₄ (pH 7.0). The columnwas washed with >10 volumes of 10 mM NaPO₄ (pH 7.0) at a flow rate of 5ml/minute. CT-CRM_(E29H) holotoxin was eluted with four column volumesof 10 mM NaPO₄ (pH 8.3). Purified CT-CRMs were buffer exchanged bydialysis into PBS and stored at 4° C. The presence of intact holotoxinand the respective subunits was determined by native polyacrylamide gelelectrophoresis (PAGE) and SDS-PAGE, respectively. Native PAGE indicatedthe presence of a purified molecule of 86 kDa (data not shown), theexpected molecular weight for intact cholera holotoxin (Tebbey et al,2000 Vaccine, 18(24):2723-2734). In addition, SDS-PAGE showed two bandsthat aligned with the CT-A (27 kDa) and CT-B (12 kDa) subunits thatcomprise the intact holotoxin (data not shown).

EXAMPLE 2 Non-Denaturing Polyacrylamide Gel Electrophoresis

[0152] Mutant CT-CRMS, CT-CRM_(R25W), CT-CRM_(R25G,) CT-CRM_(T48TH,)CT-CRM_(G34GGP) and CT-CRM_(Y30WAH), were analyzed by non-denaturingpage electrophoresis to determine the percentage of the CT-CRMs presentafter purification as intact holotoxin. Purified CT-CRMs, 15 μl each (atvarious protein concentrations), were run through a 6% polymerizednon-denaturing polyacrylamide gel. Three different concentrations (300,600 and 1200 ng) of CT-B were used as a standard. After electrophoresisthe gel was stained with Coomassie blue. The gel was then scanned usinga densitometer, and the percentage of the holotoxin was calculated fromthe densitometer readings of the CT-CRMs and CT-B standard. The dataindicated that 95% of CT-CRM_(R25W), 91.20% of CT-CRM_(R25G,) 91.00% ofCT-CRM_(T48TH,) 98.80% of CT-CRM_(G34GGP) and 90.93% of CT-CRM_(Y30WAH)were present as intact holotoxins (Table 2). TABLE 2 Native Gel Assayfor Intact Holotoxin CT-CRM % of holotoxin CT-CRM_(R25W) >95CT-CRM_(R25G) 91.0 CT-CRM_(T48TH) 91.20 CT-CRM_(G34GGP) 98.80CT-CRM_(Y30WAH) 90.93

EXAMPLE 3 Y-1 Adrenal Cell Assay for Residual Toxicity of CT-CRMs

[0153] Mutant CT-CRMs were compared with wild-type CT for toxicity inthe mouse Y-1 adrenal tumor cell assay, which is used in vitro tomeasure toxicity of enterotoxins in the cholera toxin/heat labileenterotoxin family. The assay depends upon binding of the toxin to cellsurface receptors, and the subsequent entry of the A1 subunit of thetoxin into the cytoplasm of the cell.

[0154] Native cholera toxin isolated from V. cholerae is proteolyticallynicked at the A1-A2 junction, resulting in the A1 and A2 subunits beingheld together by only a disulfide bond. This makes the A1 and A2subunits unstable and easily dissociable from each other. The A1 subunitof the nicked CT dissociates from the A2 subunit upon binding to thecell surface receptor, and enters the cell, where it ADP-ribosylates theregulatory G-protein (Gsα), leading to its toxic effects as describedabove. In contrast, enterotoxin produced in E. coli (either CT or LT) isunnicked, and thus, has the A1-A2 peptides still joined. Consequently,the CT produced in V. cholerae is significantly more toxic in the Y-1adrenal cell assays than the CT produced in a heterologous bacterialcell such as E. coli.

[0155] In a first Y-1 adrenal cell assay, mutant CT-CRMs were comparedto nicked wild-type CT from V. cholerae for toxicity. In this assay, Y-1adrenal cells (ATCC CCL-79) were seeded in 96-well flat-bottom plates ata concentration of 10⁴ cells per well. Thereafter, three-fold serialdilutions of purified (˜90% purity as determined by Coomassie staining)CT-CRMs were added to the tumor cells and incubated at 37° C. (5% CO₂)for 18 hours. The cells were then examined by light microscopy forevidence of toxicity (cell rounding). The endpoint titer was defined asthe minimum concentration of toxin required to give greater than 50%cell rounding. The percent of residual toxicity was then calculatedusing the endpoint titer of wild-type nicked CT from V. cholerae (100%toxicity) divided by the titer elicited by CT-CRMs multiplied by 100.The data set forth in Table 3 indicate that the residual toxicity of thefive purified mutant holotoxins, CT-CRM_(R25W), CT-CRM_(R25G),CT-CRM_(T48TH), CT-CRM_(G34GGP) and CT-CRM_(Y30WAH) tested using the Y-1adrenal cell assay was substantially reduced. TABLE 3 Y-1 Adrenal CellAssay CT-CRM % Residual Toxicity CT-CRM_(R25W) 0.37 CT-CRM_(R25G) 0.041CT-CRM_(T48TH) 0.12 CT-CRM_(G34GGP) 1.11 CT-CRM_(Y30WAH) 0.12

[0156] In a second independent study, crude periplasmic extracts of E.coli cells (TG1) expressing elevated levels of mutant CT-CRMs, werecompared against unnicked wild-type CT holotoxin expressed in E. colifor residual toxicity in Y-1 adrenal cell assay. Y-1 adrenal cells wereincubated in multi-well dishes in an RPMI medium containing 10% fetalcalf serum in the presence of crude E. coli cell lysate. Cell toxicitywas monitored as before. In this study, one toxic unit was defined asthe smallest amount of toxin or supernatant that caused rounding of75-100% of the cells in a well after overnight incubation. The resultsof this study are presented in Table 4 below. TABLE 4 Y-1 Adrenal CellAssay CT-CRM % Residual Toxicity CT-CRM_(R25W) 30 CT-CRM_(R25G) 6CT-CRM_(T48TH) 25 CT-CRM_(G34GGP) 30 CT-CRM_(Y30WAH) 8

[0157] The results of this study indicated that while the toxicities ofCT-CRM_(R25G) and CT-CRM_(Y30WAH) were substantially reduced, thetoxicities of CT-CRM_(R25W) and CT-CRM_(T48TH) were approximately 30% ofthe toxicity of wild-type CT. Without being bound by theory, the variantresults in the second study (Table 4) may be attributable to the factthat periplasmic crude E. coli cell lysates used in the second studycontained unnicked mutant CT-CRMs. Another contributing factor may bethat toxicity was measured as a percentage of the toxicity of wild-type,unnicked CT produced by E. coli, wherein the unnicked wild-type CT fromE. coli had a 50% cell rounding dose of 6250 pg/ml in the same Y1 cellassay (data not shown). In contrast, in the first study, the residualcytotoxicity of the mutant CT-CRMs is expressed as a percentage of thetoxicity of wild-type, nicked CT produced by V. cholerae, wherein thenicked holotoxin had a 50% cell rounding dose of 125 pg/ml in the sameY1 cell assay. Consequently, the residual toxicity reported in thesecond study is 50 fold higher than that obtained in the first study.

EXAMPLE 4 The ADP-Ribosyltransferase Assay

[0158] NAD⁺:agmatine ADP-ribosyltransferase activity was measured as therelease of [carbonyl-¹⁴C] nicotinamide from radiolabeled NAD⁺. Briefly,CT and CT-CRMs were trypsin activated and incubated for 30 minutes at30° C. with 50 mM glycine/20 mM dithiothreitol in TEAN buffer(Tris/EDTA/sodium azide/sodium chloride) (pH 8.0). Thereafter, thefollowing materials were added to the reaction: 0.1 μg of soybeantrypsin inhibitor, 50 mM potassium phosphate, 10 mM agmatine, 20 mMdithiothreitol, 10 mM magnesium chloride, 100 μM GTP, 3 mMdimyristoylphosphatidyl-choline, 0.2% cholate, 0.03 mg of ovalbumin, 100μM [adenine-U-¹⁴C]NAD (DuPont NEN™, Boston, Mass.) and water to a finalvolume of 300 μl. After incubation for 90 minutes at 30° C., 100 μlsamples were applied to columns (0.64×5 cm) of AG1-X2 (Bio-Rad), whichwere washed five times with 1.0 ml of distilled/deionized H₂O. Eluatescontaining [¹⁴C]ADP-ribosylagmatine were collected for radioassay. Meanrecovery of ¹⁴C in the eluate is expressed as percentage of that appliedto column. The results are presented in Table 5. TABLE 5 NAD:AgmatineADP-Ribosyltransferase Activity ADP-ribosylagmatine % ADP- formedribosylation CT/CT-CRM (nmol/hr/μg protein) activity CT, 10 μg 35.7 100CT-CRM_(R25W) 1.6 4.5 CT-CRM_(R25G) 1.0 2.7 CT-CRM_(T48TH) 1.2 3.4CT-CRM_(G34GGP) 1.8 5.0 CT-CRM_(Y30WAH) 1.6 4.5

[0159] ADP-ribosyltransferase activity was also independently determinedusing diethylamino (benzylidine-amino) guanidine (DEABAG) as a substrate(Jobling, MG and Holmes, R K 2001 J. Bacteriol., 183(13):4024-32). Inthis assay, 25 μl aliquots of mutant CT-CRMs from purified cell lysates,activated for 30 minutes at 30° C. with 1/50 w/w trypsin, were incubatedwith 200 μl 2 mM DEABAG in 0.1M K₂PO₄, pH 7.5, 10 μM NAD, 4 mM DTT fortwo hours. The reaction was stopped by adding 800 μl of a slurry buffercontaining 400 mg DOWEX AG50-X8 resin to bind unreacted substrate.ADP-ribosylated DEABAG in the supernatant was quantitated by florescenceemission in a DyNA Quant fluorimeter calibrated with DEABAG. With theexception of the mutants CT-CRM_(G34GGP) and CT-CRM_(Y30WAH), the ADPribosyl-transferase activities of the mutant CT-CRMs were substantiallyreduced over that of wild-type (Table 6). The high level ofADP-ribosyl-transferase activity seen with CT-CRM_(G34GGP) andCT-CRM_(Y30WAH) may be attributable to the fact that in this study theADP ribosyl-transferase activity of mutant CT-CRMs was measured using adifferent substrate in a different assay protocol. TABLE 6ADP-ribosyltransferase Activity of CT-CRMs using Diethylamino(benzylidine-amino) Guanidine (DEABAG) CT/CT-CRM % ADP-ribosylationActivity CT 100 CT-CRM_(R25W) 10 CT-CRM_(R25G) 0.5 CT-CRM_(T48TH) 18CT-CRM_(G34GGP) 54 CT-CRM_(Y30WAH) 43

EXAMPLE 5 Immune Responses of BALB/C Mice Immunized with Recombinant P4Outer Membrane Protein (rP4) of Nontypable Haemophilus influenzae (NTHi)Alone or in Conjunction with CT-CRMS

[0160] BALB/c mice (6-8 weeks old, 5 mice/group) were immunized at weeks0, 3 and 5 with recombinant P4 protein (rP4, 5 μg per dose) in saline orco-formulated with wild-type CT, CT-CRM_(E29H), CT-CRM_(T48TH),CT-CRM_(G34GGP), CT-CRM_(Y30WAH), CT-CRM_(R25W) or CT-CRM_(R25G) at adose 1.0 μg per immunization. A total volume of 10 μl was administeredintranasally (5 μl per nostril). Mice were bled at weeks 0, 2, 3, 4, 5or 6 in order to assay serum antibody responses. One week after the lastimmunization (week 6), mice were sacrificed for the analysis of mucosalantibody responses.

[0161] Significant differences between groups were determined by theTukey-Kramer HSD multiple comparisons test (for rP4 protein) or by theStudent t-test (for UspA2) using JMP® statistical discovery software(SAS Institute Inc., Cary, N.C.).

[0162] Analysis of serum antibodies at weeks 0, 3, 5 and 6 showed thatimmunization with NTHi rP4 protein formulated with any of the CT-CRMmutants, disclosed herein at a concentration of 1 μg/dose, significantlyinduced immune responses to rP4 protein. The magnitude of the total IgGimmune response to rP4 protein was increased approximately 15-35 fold byinclusion of the CT-CRM mutants in the formulation. No significantdifferences were observed in total anti-rP4 IgG titers among the newmutant toxins (CT-CRM_(T48TH), CT-CRM_(G34GGP), CT-CRM_(Y30WAH),CT-CRM_(R25W) and CT-CRM_(R25G)) even though they all elicitedsignificantly higher IgG titers than rP4 protein alone by Student t-test(Table 6). Individual serum analysis of IgA antibodies showed that onlyformulation rP4/CT-CRM_(R25G) elicited significantly higher titers ofIgA antibodies to rP4 protein than the control group receiving rP4protein in saline (Table 7). The use of each of the new CT-CRM mutantsalso enhanced serum IgG subclass antibodies (IgG1, IgG2a and IgG2b) torP4 protein (Table 9).

[0163] Anti-rP4 protein antibody responses were also analyzed in pooledmucosal wash samples (Table 8). As expected, no induction of antibody inBAL and NW from rP4/saline immunized mice was observed. However, thepotent mucosal adjuvant capacity of CT-CRM_(T48TH), CT-CRM_(Y30WAH) andCT-CRM_(R25G) was clearly demonstrable. Although no statistical analysiscan be performed on these pooled samples, some trends appeared. Forexample, mice that received CT-CRM_(T48TH), CT-CRM_(Y30WAH) andCT-CRM_(R25G) displayed elevated rP4 specific IgA antibodies in each ofthe saliva, NW and VW samples tested.

[0164] Additionally, anti-CT antibody responses were also determined. Asshown in Tables 7-11, all the CT mutants enhanced the systemic andmucosal CT-specific antibody responses one-week after the lastimmunization. However, it should be noted that the mice in the abovestudy were, subsequent to the completion of the study, determined to beinfected with the mouse hepatitis virus. TABLE 7 The Effect of MutantCholera Toxins on the Immunogenicity of NTHi LrP4 Protein followingIntranasal Immunization in BALB/c Mice. Anti-rLP4 Antibody Titers^(c)(Pooled Sera)^(d) Week 0 Week 3 Week 5 Week 6 Immunogen Rte^(a)Adjuvant^(b) IGA^(b) IgG IgA IgG IgA IgG IgA IgG NTHi LrP4 IN — <100<100 <100 168 <100 532 122 3,638 NTHi LrP4 IN CT <100 120 <100 795 19818,584 NTHi LrP4 IN CT-CRM_(E29H) <100 <100 <100 576 152 4,854 NTHi LrP4IN CT-CRM_(T48TH) <100 102 111 10,594 453 55,775 NTHi LrP4 INCT-CRM_(G34GGP) <100 146 196 1,701 434 68.325 NTHi LrP4 INCT-CRM_(Y30WAH) <100 <100 <100 3,406 391 93,502 NTHi LrP4 INCT-CRM_(R25W) <100 187 116 16,809 509 127,130 NTHi LrP4 IN CT-CRM_(R25G)<100 278 273 23,163 1,056 62,323

[0165] TABLE 8 The Effect of Mutant Cholera Toxins on the Immunogenicityof NTHi LrP4 Protein following Intranasal Immunization in BALB/c Mice.Anti-rLP4 Antibody Titers^(c) (Pooled Mucosal Washes)^(d) SAL BAL VW NWImmunogen Rte^(a) Adjuvant^(b) IgA IgG IgA IgG IgA IgG IgA IgG NTHi LrP4IN — 19 <10 <10 <10 33 <10 <10 <10 NTHi LrP4 IN CT <10 <10 <10 23 <10<10 <10 <10 NTHi LrP4 IN CT-CRM _(E29H) <10 <10 <10 <10 33 <10 <10 <10NTHi LrP4 IN CT-CRM _(T48TH) 105 <10 <10 79 73 21 12 <10 NTHi LrP4 INCT-CRM _(G34GGP) 17 <10 <10 80 11 23 <10 13 NTHi LrP4 IN CT-CRM_(Y30WAH) 25 <10 <10 113 48 47 10 19 NTHi LrP4 IN CT-CRM _(R25W) 37 <10<10 169 20 23 <10 <10 NTHi LrP4 IN CT-CRM _(R25G) 185 <10 <10 64 348 3223 <10

[0166] TABLE 9 The Effect of Mutant Cholera Toxins on the Immunogenicityof NTHi LrP4 Protein following Intranasal Immunization in BALB/c Mice.Anti-rLP4 Antibody Titers^(c) (Week 6 Pooled Sera)^(d) Immunogen Rte^(a)Adjuvant^(b) IgG1 IgG2a IgG2b IgG3 NTHi LrP4 IN — 1,165 1,891 1,245 <100NTHi LrP4 IN CT 625 14,989 9,284 278 NTHi LrP4 IN CT- 1,630 2,618 845<100 CRM_(E29H) NTHi LrP4 IN CT- 11,220 35,239 20,733 206 CRM_(T48TH)NTHi LrP4 IN CT- 12,583 48,134 24,267 <100 CRM_(G34GGP) NTHi LrP4 IN CT-13,894 59,049 28,975 744 CRM_(Y30WAH) NTHi LrP4 IN CT- 24,373 89,89237,389 422 CRM_(R25W) NTHi LrP4 IN CT- 7,957 46,776 16,731 256CRM_(R25G)

[0167] TABLE 10 The Effect of Mutant Cholera Toxins on theImmunogenicity of NTHi LrP4 Protein following Intranasal Immunization inBALB/c Mice. Anti-rLP4 Antibody Titers on Individual Sera^(c,d)Immunogen Rte^(a) Adjuvant^(b) 1 2 3 4 5 GeoMean^(c) StDev NTHi LrP4 IN— 35 114 61 79 316 121 113 NTHi LrP4 IN CT 139 48 145 85 461 176 165NTHi LrP4 IN CT-CRM_(E29H) 33 126 333 26 49 113{circumflex over ( )} 129NTHi LrP4 IN CT-CRM_(T48TH) 468 780 530 76 218 414 275 NTHi LrP4 INCT-CRM_(G34GGP) 177 479 963 175 214 402 338 NTHi LrP4 IN CT-CRM_(Y30WAH)271 443 408 699 282 421 173 NTHi LrP4 IN CT-CRM_(R25W) 431 198 361 360835 437 238 NTHi LrP4 IN CT-CRM_(R25G) 1,037 462 1,851 1,825 6781,171*{circumflex over ( )}  643

[0168] TABLE 11 The Effect of Mutant Cholera Toxins on theImmunogenicity of NTHi LrP4 Protein following Intranasal Immunization inBALB/c Mice. Anti-rLP4 Antibody Titers on Individual Sera^(c,d)Immunogen Rte^(a) Adjuvant^(b) 1 2 3 4 5 GeoMean^(e) StDev NTHi LrP4 IN— 112 2,393 2,885 4,432 8,471 3,659 3,104 NTHi LrP4 IN CT 22,042 5,49913,292 10,746 24,920 15,300  8,044 NTHi LrP4 IN CT-CRM_(E29H) 3,06316,889 179 204 1,406 4,348{circumflex over ( )} 7,109 NTHi LrP4 INCT-CRM_(T48TH) 63,090 52,393 53,912 13,017 38,604 44,203* 19,506 NTHiLrP4 IN CT-CRM_(G34GGP) 63,924 63,908 73,793 59,169 50,229 62,205* 8,555NTHi LrP4 IN CT-CRM_(Y30WAH) 63,522 65,791 31,934 174,833 130,17393,251* 57,915 NTHi LrP4 IN CT-CRM_(R25W) 138,982 88,085 160,963 74,885151,979 122,979*  38,957 NTHi LrP4 IN CT-CRM_(R25G) 47,114 15,915154,578 34,780 17,598 54,003* 57,680

EXAMPLE 6 The Immune Responses of BALB/C Mice Immunized with the UspA2Outer Membrane Protein of M. catarrhalis

[0169] In this study, the capacity of mutant CT-CRMs to augment systemicand mucosal immune responses against the native UspA2 outer membraneprotein of M. catarrhalis was examined. BALB/C mice (6-8 weeks old, 5mice/group) were immunized at weeks 0, 3, and 5. Purified UspA2 (5μg/dose) alone in 10 μl saline or in a 10 μl formulation containing 0.1μg/dose of wild-type CT or a mutant CT-CRM (CT-CRM_(E29H),CT-CRM_(R25W), CT-CRM_(R25G), CT-CRM_(T48TH,) CT-CRM_(G34GGP), orCT-CRM_(Y30WAH)) was administered to Balb/C mice IN (5 ul/nostril) onweek 0, 2 and 4. Analysis of serum antibodies at weeks 0, 2, 4 and 6showed that immunization with UspA2 formulated with any of theaforementioned CT-CRM mutants except CT-CRM_(R25G), at a concentrationof 0.1 μg/dose, enhanced antibody responses to UspA2 (Table 12). Themagnitude of the total IgG immune response to UspA2 was increasedapproximately 3-10 fold by inclusion of the CT-derived mutants.CT-CRM_(G34GGP), CT-CRM_(E29H) or wild-type CT elicited significantlyhigher IgG titers than UspA2/PBS by Student t-test. However, nosignificant differences were observed in total anti-UspA2 IgG titersbetween each of the new mutant toxins (CT-CRM_(E29H), CT-CRM_(T48TH),CT-CRM_(G34GGP), CT-CRM_(Y30WAH), CT-CRM_(R25W), or CT-CRM_(R25G)). Theuse of each of the CT mutants except CT-CRM_(R25W), also enhanced serumIgG1, IgG2a and IgG2b antibodies to UspA2 (Table 12).

[0170] Anti-UspA2 antibody responses were also analyzed in pooledmucosal wash samples (Table 13). As expected, no induction of antibodyin mucosal washes from UspA2/PBS immunized mice was observed. However,the potent mucosal adjuvant capacity of each mutant CT was clearlydemonstrated. Although no statistical analyses were performed on thesepooled samples, some trends emerged. For example, mice that receivedCT-CRM_(G34GGP) displayed elevated IgG or IgA titers to UspA2 in each ofthe bronchoalveolar lavage (BAL), nasal wash (NW) and vaginal wash (VW)samples collected (Table 13). In comparison, none of the new mutanttoxins appeared to be better than CT-CRM_(E29H) or wild-type CT inadjuvanting local immune responses to UspA2 protein. Protein-specificIgG and IgA levels in the serum and in mucosal lavages were alsoexamined on day 28. All mutant CTs elicited enhanced serum IgG antibodyresponse (data not shown). The levels of IgG and IgA in bronchial, nasaland vaginal washes were also measured. No IgA was detected in any of thewashes, and IgG was detected only in a few washes (Table 13). The micein this study were, subsequent to the completion of the study,determined to be infected with the mouse hepatitis virus. TABLE 12 SeraIgG subclass titers against UspA2 Week 6 * IgG1/G2a Antigen AdjuvantIgG1 IgG2a IgG2b IgG3 Ratio UspA2 None 600 346 320 <100 1.7 UspA2 CT-3,443 6,655 4,027 <100 0.52 CRM_(E29H) UspA2 CT- 1,350 809 429 <100 1.6CRM_(T48TH) UspA2 CT- 5,918 4,480 3,111 <100 1.32 CRM_(G34GGP) UspA2 CT-1,991 1,618 809 <100 1.23 CRM_(Y30WAH) UspA2 CT- 1,772 1,100 1,095 <1001.61 CRM_(R25W) UspA2 CT- 301 221 224 <100 1.36 CRM_(R25G) UspA2 CT9,050 18,227 10,195 189 0.5

[0171] TABLE 13 Mucosal IgG & IgA titers against UspA2 * BW NW VWAntigen Adjuvant IgG IgA IgG IgA IgG IgA UspA2 None <10 <10 <10 <10 <10<10 UspA2 CT-CRM_(E29H) 16 <10 <10 20 24 506 UspA2 CT-CRM_(T48TH) <10<10 <10 <10 <10 58 UspA2 CT-CRM_(G34GGP) 17 <10 <10 31 24 285 UspA2CT-CRM_(Y30WAH) <10 <10 <10 32 <10 208 UspA2 CT-CRM_(R25W) <10 <10 <1020 <10 29 UspA2 CT-CRM_(R25G) <10 <10 <10 <10 <10 <10 UspA2 CT 40 <10<10 85 111 774

EXAMPLE 7 Adjuvanticity of the Mutant Cholera Toxin Holotoxins

[0172] To create a comprehensive panel of mutant CT-CRMs with differentcharacteristics of toxicity, functionality and immunogenicity, theabove-described CT-CRM mutants were analyzed as mucosal adjuvants, andthe toxicity and enzymatic activity profiles of each of the mutants weredetermined. As summarized in Tables 14 through 18, all mutant CT-CRMshave significantly reduced toxicity and enzyme activity compared towild-type CT. These genetically detoxified mutant CTs were evaluated fortheir capacity to adjuvant immune responses to native UspA2 protein fromM. catarrhalis.

[0173] The experiments were performed as follows: BALB/c mice (6-8 weeksold, 5 mice/group) were immunized at weeks 0, 2 and 4 with 5 μg ofpurified native UspA2 protein in PBS or co-formulated with doses of 0.1or 1.0 μg per immunization of wild-type CT, or CT-CRM_(E29H), orCT-CRM_(T48TH), or CT-CRM_(G34GGP), or CT-CRM_(Y30WAH), or CT-CRM_(R24W)or CT-CRM_(R25G). A total volume of 10 μl was administered intranasally(5 μl per nostril). Mice were bled at weeks 0, 2, 4, or 6 in order toassay serum antibody responses. Two weeks after the last immunization(week 6), mice were sacrificed for the analysis of mucosal antibodyresponses. UspA2 ELISA titers were determined at an endpoint of 0.1 atOD₄₀₅. Significant differences between groups were determined by theTukey-Kramer HSD multiple comparisons test using JMP® statisticaldiscovery software (SAS Institute Inc., Cary, N.C.).

[0174] Adjuvanticity of the CT-CRMs can be summarized as follows.Analysis of serum IgG and IgA antibodies at weeks 2, 4 and 6 showed thatimmunization with UspA2 protein formulated with any of the CT-CRMmutants, except CT-CRM_(R25G), at a concentration of 1 μg/dose,significantly enhanced antibody responses to UspA2 protein (Table 15).The magnitude of the total IgG immune response to UspA2 protein wasincreased approximately 11-68 fold by inclusion of the CT-derivedmutants (excluding CT-CRM_(R25G)) (Table 16). CT-CRM_(T48TH),CT-CRM_(Y30WAH), CT-CRM_(R25W) (at 1 μg dose), and CT-CRM_(G34GGP) (atboth 0.1 μg and 1 μg doses) elicited significantly higher IgG and IgAtiters than UspA2/PBS by Tukey-Kramer analysis. However, no significantdifferences were observed in total anti-UspA2 IgG titers between each ofthe new mutant toxins excluding CT-CRM_(R25G) (Table 16). The use ofeach of the CT mutants except CT-CRM_(R25G) at a 1 μg dose also enhancedserum IgG1, IgG2a and IgG2b antibodies to UspA2 (Table 17). The ratio ofthe IgG1 and IgG2a/IgG2b titers was approximately 1.0, indicating abalanced Th1/Th2 type of immune response.

[0175] Anti-UspA2 protein antibody responses were also analyzed inpooled mucosal wash samples (Table 18). As expected, no induction ofantibody in mucosal washes from UspA2/PBS immunized mice was observed.However, the potent mucosal adjuvant capacity of each mutant CT-CRM,excluding CT-CRM_(R25G) was clearly demonstrated. There were UspA2specific mucosal IgA antibodies detected in most of the mucosal samples.Although no statistical analysis can be performed on these pooledsamples, some trends appeared. For example, mice that receivedCT-CRM_(G34GGP) or CT-CRM_(R25W) displayed elevated IgG or IgAantibodies to UspA2 protein in each of the bronchoalveolar lavage, thenasal wash and the vaginal wash samples collected, similar to thewild-type CT or CT-CRM_(E29H).

[0176] These CT-CRMs, except CT-CRM_(R25G), are potent mucosal adjuvantsfor M. catarrhalis UspA2 protein. The serum antibody data showed thatall the CT-CRMs except CT-CRM_(R25G) at 1 μg dose are equally as capablein adjuvanting immune responses to UspA2 protein as is CT-CRM_(E29H)(Table 16). At 0.1 μg of dose, CT-CRM_(G34GGP) appeared to be morepotent than CT-CRM_(E29H) at the same dose (Table 16). The mucosal washdata appears to suggest that all of these mutant CT-CRMs exceptCT-CRM_(R25G), retain potent mucosal adjuvant properties (Table 18).Furthermore, they all have significantly lower residual toxicity andenzyme activity than wild-type CT as shown in Table 14. Therefore, thesemutant CT-CRMs are additional effective mucosal adjuvants. TABLE 14Characterization of the Mutant Cholera Toxins Y-1 cell ADP-Ribosyl-Homoge- Holotoxin toxicity transferase Mutant CT neity (%) (%) (%)activity (%) CT-CRM_(T48TH) 100.0 91.0 0.12 3.4 CT-CRM_(G34GGP) 100.098.8 1.11 5.0 CT-CRM_(Y30WAH) 99.0 90.9 0.12 4.5 CT-CRM_(R25W)100.0 >95.0 0.37 4.5 CT-CRM_(R25G) 99.7 91.2 0.041 2.7

[0177] TABLE 15 Adjuvant Effects of Mutant CT on the Immune Response toUspA2 Delivered IN to Female BALB/c Mice Antigen week 2 week 4 week 6 (5μg) Adjuvant Dose IgG IgA IgG IgA IgG IgA UspA2 None PBS <100 <100 <100<100 <500 <50 UspA2 CT-CRM_(E29H) 1 μg 182 54 3,777 <100 11,305 194 0.1μg <100 <100 160 <100 544 <50 UspA2 CT-CRM_(T48TH) 1 μg <100 <100 967<100 3,542 128 0.1 μg <100 <100 <100 <100 568 <50 UspA2 CT-CRM_(G34GGP)1 μg 298 <100 6,170 83 15,498 398 0.1 μg 125 <100 775 <100 2,900 98UspA2 CT-CRM_(Y30WAH) 1 μg 206 <100 1,275 <100 3,330 81 0.1 μg <100 <100<100 <100 <500 <50 UspA2 CT-CRM_(R25W) 1 μg 304 <100 8,335 <100 16,308196 0.1 μg <100 <100 214 <100 989 <50 UspA2 CT-CRM_(R25G) 1 μg <100 <100232 <100 <1,000 <50 0.1 μg <100 <100 <100 <100 <500 <50 UspA2 CholeraToxin 1 μg 191 <100 6,119 <100 13,588 254 0.1 μg 351 <100 5,472 10820,632 399

[0178] BALB/c mice (groups of 5) were immunized IN with a 10 ul volumeat weeks 0, 2, & 4. Sera were collected at week 6. The UspA2 ELISAtiters were determined at an endpoint of 0.1 at OD₄₀₅. The Tukey-Krameranalysis showed the following: The 1 μg dose of each adjuvant isstatistically significant from the same adjuvant at 0.1 g dose, exceptthe IgG of CT, CT-CRM_(G34GGP) and IgA of CT-CRM_(R25G). Results inTable 16 reported with an asterisk (*) are statistically significantfrom the UspA2/PBS group. Results indicated with footnote a (^(a)) arestatistically significantly higher than all 0.1 μg doses (except CT) andthe 1 μg dose of CT-CRM_(R25G). Results indicated with footnote b (^(b))are statistically significantly lower than all 1 μg doses except the 1μg dose of CT-CRM_(T48TH). Results indicated with footnote c (^(c)) arestatistically significantly higher than all 0.1 μg doses (except CT) andthe 1 μg dose of CT-CRM_(R25G). Results indicated with a footnote d(^(d)) are statistically significantly lower than all 1 μg doses andalso the 0.1 μg dose of CT and CT-CRM_(G34GGP). TABLE 16 IndividualSerum Analysis of IgG and IgA Titers against UspA2 Serum Anti-UspA2Protein Antibody Titers (Mean Antigen Log₁₀) (5 μg) Adjuvant Dose IgGIgA UspA2 PBS — 2.21 ± 0.36  <25 UspA2 CT-CRM_(E29H) 1 μg 4.33 ± 0.19*2.59 ± 0.20* 0.1 μg   2.68 ± 0.34  1.16 ± 0.13  UspA2 CT- 1 μg 3.74 ±0.45* 2.20 ± 0.66* CRM_(T48TH) 0.1 μg   2.51 ± 0.56  <25 UspA2 CT- 1 μg4.53 ± 0.11* 2.76 ± 0.15* CRM_(G34GGP) 0.1 μg     3.95 ± 0.20^(a)*  2.16 ± 0.27*^(c) UspA2 CT- 1 μg 3.84 ± 0.30* 2.03 ± 0.34* CRM_(Y30WAH)0.1 μg   2.05 ± 0.56  <25 UspA2 CT-CRM_(R25W) 1 μg 4.52 ± 0.46* 2.52 ±0.25* 0.1 μg   3.25 ± 0.39* 1.28 ± 0.26  UspA2 CT-CRM_(R25G) 1 μg  2.96± 0.33^(b)   1.53 ± 0.35^(d)  0.1 μg   1.89 ± 0.27  1.20 ± 0.23  UspA2Cholera 1 μg 4.61 ± 0.15* 2.69 ± 0.31* Toxin 0.1 μg   4.44 ± 0.24* 2.89± 0.23*

[0179] The data reported in Table 17 was based upon the followingexperiment.

[0180] Groups of five female BALB/c mice were immunized intranasally atweeks 0, 2, and 4 with 10 μL containing 5 μg nUspA2 adjuvanted with hugCT (Sigma) or CT mutants. Endpoint antibody titers were determined fromsera collected at week 6. Data are presented in Table 17 as thegeometric mean (±1 SD) of the reciprocal dilution resulting in an OD₄₀₅of 0.1. Statistical analysis by Tukey-Kramer indicated that resultsmarked with an asterisk (*) were significantly higher than thenUspA2/PBS group. TABLE 17 The serum anti-nUspA2 responses of BALB/cmice after intranasal immunization with nUspA2 adjuvanted with mutantCTs Antigen Mean log 10 Antibody Titers (± 1SD) Group (5 μg) AdjuvantIgG1 IgG2a IgG2b AG673 nUspA2 PBS <2.00 <2.00 <2.00 AG674 nUspA2CT-CRM_(E29H) (1 μg) 3.14 ± 0.23* 3.44 ± 0.41* 2.92 ± 0.20 AG676 nUspA2CT-CRM_(T48TH) (1 μg) 2.45 ± 0.31 2.85 ± 0.52* 2.47 ± 0.33 AG678 nUspA2CT-CRM_(G34GGP) (1 μg) 3.06 ± 0.16* 3.55 ± 0.09* 3.00 ± 0.02 AG680nUspA2 CT-CRM_(Y30WAH) (1 μg) 2.61 ± 0.28* 2.77 ± 0.23* 2.37 ± 0.27AG682 nUspA2 CT-CRM_(R25W) (1 μg) 3.29 ± 0.40* 3.43 ± 0.57* 3.07 ± 0.30AG684 nUspA2 CT-CRM_(R25G) (1 μg) 2.11 ± 0.18 2.05 ± 0.09 <2.00 AG686nUspA2 CT (1 μg) 3.14 ± 0.28* 3.39 ± 0.27* 3.24 ± 0.29

[0181] For the data in Table 18, BALB/c mice (5/group) were immunized INwith a 10 μl volume at weeks 0, 2 and 4. Mucosal wash samples werecollected at week 6. UspA2 ELISA titers were determined at an endpointof 0.1 at OD₄₀₅. TABLE 18 UspA2 ELISA - Mucosal Antibody Titers AntigenBronch washes Nasal washes Vaginal washes (5 μg) Adjuvant Dose IgG IgAIgG IgA IgG IgA UspA2 — — <10 <10 <10 <10 <10 <10 UspA2 CT-CRM_(E29H) 1μg 21 <10 <10 17 54 500 0.1 μg <10 <10 <10 <10 <10 <10 UspA2CT-CRM_(T48TH) 1 μg <10 <10 <10 <10 <10 293 0.1 μg <10 <10 <10 <10 <10<10 UspA2 CT-CRM_(G34GGP) 1 μg 22 <10 <10 12 46 1,103 0.1 μg <10 <10 <1018 <10 617 UspA2 CT-CRM_(Y30WAH) 1 μg 11 <10 <10 <10 12 105 0.1 μg <10<10 <10 <10 <10 <10 UspA2 CT-CRM_(R25W) 1 μg 24 <10 <10 24 <10 323 0.1μg <10 <10 <10 <10 <10 13 UspA2 CT-CRM_(R25G) 1 μg <10 <10 <10 <10 <10<10 0.1 μg <10 <10 <10 <10 <10 <10 UspA2 Cholera Toxin 1 μg 41 24 <10 1419 990 0.1 μg 24 10 <10 47 41 460

EXAMPLE 8 The Immune Responses of BALB/c Mice Immunized with thePurified Native Fusion (F) Protein of Respiratory Syncytial Virus (RSV)

[0182] The capacity of the mutant CT-CRMs of the present invention toaugment the mucosal immune responses against respiratory syncytial virus(RSV) proteins was examined using the purified native fusion (F)protein.

[0183] Naïve BALB/c mice (8-10 weeks of age, 5/group) were immunized(IN, 10 μl) at weeks 0 and 3 with native purified fusion (F) proteinpurified from the 248/404 strain of RSV. The protein (3 μg/dose) wasprepared in mixture with 1.0 or 0.1 μg of the indicated CT-CRM. Controlmice were immunized with F protein admixed with CT-CRM_(E29H) alone,with wild-type CT, or with PBS. Serum (geometric mean titer±1 standarddeviation) and bronchoalveolar (BAW), nasal (NW) and vaginal (VW) washfluids were collected two weeks after secondary immunization for thedetermination of end-point anti-F protein total and subclass IgG and IgAtiters by ELISA. The mucosal wash samples were pooled for thedetermination of endpoint titers.

[0184] The results from two experiments are presented in Tables 19 and20. TABLE 19 The Humoral Immune Response to BALB/c Mice after IntranasalImmunization with F Protein and CT-CRMs Geometric Mean Serum Anti-FProtein Ig Titers (Log₁₀) Antigen Adjuvant (μg) IgG IgG1 IgG2a IgA Fprotein NONE 2.6 ± 1.5 2.3 ± 1.3 2.0 ± 0.8 <1.7 F proteinCT-CRM_(T48TH)(1) 5.7 ± 0.1 5.4 ± 0.2 4.5 ± 0.3 4.0 ± 0.3 F proteinCT-CRM_(T48TH)(0.1) 4.3 ± 1.0 4.4 ± 1.0 3.5 ± 0.5 2.7 ± 0.9 F proteinCT-CRM_(G34GCP)(1) 5.8 ± 0.3 5.3 ± 0.2 4.9 ± 0.4 4.2 ± 0.2 F proteinCT-CRM_(G34GCP)(0.1) 5.4 ± 0.2 5.0 ± 0.3 4.1 ± 0.3 4.1 ± 0.2 F proteinCT-CRM_(Y30WAH)(1) 5.9 ± 0.4 5.1 ± 0.2 4.5 ± 0.3 3.9 ± 0.3 F proteinCT-CRM_(Y30WAH)(0.1) 4.7 ± 0.5 4.9 ± 0.4 3.6 ± 0.5 3.1 ± 0.5 F proteinCT-CRM_(R25W)(1) 6.1 ± 0.3 5.7 ± 0.3 4.5 ± 0.2 4.2 ± 0.2 F proteinCT-CRM_(R25W)(0.1) 5.5 ± 0.4 5.3 ± 0.4 4.2 ± 0.3 4.0 ± 0.1 F proteinCT-CRM_(R25G)(1) 5.4 ± 0.4 4.9 ± 0.6 4.0 ± 0.4 3.9 ± 0.2 F proteinCT-CRM_(R25G)(0.1) 3.8 ± 0.9 3.7 ± 0.8 3.0 ± 0.4 2.6 ± 0.8 F proteinCT-CRM_(E29H)(1) 5.9 ± 0.4 5.4 ± 0.4 4.8 ± 0.3 4.3 ± 0.1 F proteinCT-CRM_(E29H)(0.1) 5.9 ± 0.4 5.3 ± 0.2 4.5 ± 0.3 4.4 ± 0.3 F proteinCT(1) 5.6 ± 1.2 5.2 ± 1.1 4.5 ± 1.1 4.3 ± 0.8 F protein CT(0.1) 5.0 ±0.3 5.2 ± 0.3 4.5 ± 0.3 4.2 ± 0.2

[0185] TABLE 20 The Humoral Immune Response to BALB/c Mice afterIntranasal Immunization with F Protein and Genetically DetoxifiedMutants of CT Anti-F Protein Antibody Titers Anti- Adjuvant BAW NW VWgen (μg) IgG IgA IgG IgA IgG IgA F protein NONE <25 <25 64 <25 <25 <25 Fprotein CT-CRM_(T48TH)(1) 227 33 146 2,560 112 2,065 F proteinCT-CRM_(T48TH)(0.1) 59 27 <25 384 <25 344 F protein CT-CRM_(G34GCP)(1)964 458 60 708 125 562 F protein CT-CRM_(G34GCP)(0.1) 181 <25 117 352 57755 F protein CT-CRM_(Y30WAH)(1) 312 <25 <25 177 52 1,332 F proteinCT-CRM_(Y30WAH)0.1) 111 24 63 210 <25 139 F protein CT-CRM_(R25W)(1) 20033 34 378 35 665 F protein CT-CRM_(R25W)(0.1) 137 32 61 557 55 633 Fprotein CT-CRM_(R25G)(1) 230 42 22 283 <25 307 F proteinCT-CRM_(R25G)(0.1) 44 <25 <25 39 <25 125 F protein CT-CRM_(E29H)(1) 27759 846 932 18 832 F protein CT-CRM_(E29H)(0.1) 142 60 245 239 <25 496 Fprotein CT(1) 398 114 68 504 <25 981 F protein CT(0.1) 158 <25 <25 360189 903

[0186] When the CT-CRM mutants of this invention were used as mucosaladjuvants at the 1.0 μg dose, results similar to the use of mutantCT-CRM_(E29H) or wild-type CT were obtained (Table 19). Noteworthydifferences from the anti-F protein IgG or IgA titers elicited followingimmunization with F protein admixed with CT-CRM_(E29H) or wild-type CTwere not observed. However, at the 0.1 μg dose, CT-CRM_(T48TH),CT-CRM_(Y30WAH) and CT-CRM_(R25G) appeared less able to augment serumanti-F protein IgA titers. The titers in the mucosal wash fluids of riceimmunized with F protein formulated with the mutants of this inventionappeared comparable to those induced by F protein admixed withCT-CRM_(E29H) or wild-type CT (Table 20).

[0187] Thus, all CT-CRM mutants of this invention had adjuvant activityfor F protein.

[0188] All publications and references cited in this specification areincorporated herein by reference. While the invention has been describedwith reference to a particularly preferred embodiment, it will beappreciated that modifications can be made without departing from thespirit of the invention. Such modifications are intended to fall withinthe scope of the appended claims.

1. An immunogenic, mutant cholera holotoxin (CT-CRM) comprising an aminoacid sequence of subunit A of the wild-type cholera toxin (CT), whereinsaid subunit A comprises at least a single amino acid substitution inthe amino acid position 25 in the A subunit, and wherein the mutantCT-CRM has reduced toxicity compared to said wild-type CT.
 2. The CT-CRMaccording to claim 1, wherein the amino acid arginine in the amino acidposition 25 in the A subunit is substituted with a tryptophan.
 3. TheCT-CRM according to claim 1, wherein the amino acid arginine in theamino acid position 25 in the A subunit is substituted with a glycine.4. The CT-CRM according to claim 1, further comprising at least oneadditional mutation in the A subunit of the cholera holotoxin at anamino acid position other than the amino acid position 25 in the Asubunit.
 5. The CT-CRM according to claim 4, wherein said additionalmutation is a substitution for a subunit A amino acid selected from thegroup consisting of the arginine at amino acid position 7, the asparticacid at amino acid position 9, the arginine at amino acid position 11,the glutaric acid at position 29, the histidine at amino acid position44, the valine at amino acid position 53, the arginine at amino acidposition 54, the serine at amino acid position 61, the serine at aminoacid position 63, the histidine at amino acid position 70, the valine atamino acid position 97, the tyrosine at amino acid position 104, theproline at amino acid position 106, the histidine at amino acid position107, the serine at amino acid position 109, the glutamic acid at aminoacid position 110, the glutamic acid at amino acid position 112, theserine at amino acid position 114, the tryptophan at amino acid position127, the arginine at amino acid position 146, and the arginine at aminoacid position
 192. 6. The CT-CRM according to claim 4, wherein saidadditional mutation is selected from the group consisting of a singleamino acid insertion in the amino acid position 49 in the A subunit, adouble amino acid insertion in the amino acid positions 35 and 36 in theA subunit, a single amino acid substitution in the amino acid position30, and a double amino acid insertion in the amino acid positions 31 and32 in the A subunit.
 7. An immunogenic, mutant cholera holotoxin(CT-CRM) comprising an amino acid sequence of subunit A of the wild-typecholera toxin (CT), wherein said subunit A comprises at least a singleamino acid insertion in the amino acid position 49 in the A subunit, andwherein the mutant CT-CRM has reduced toxicity compared to saidwild-type CT.
 8. The CT-CRM according to claim 7, wherein the amino acidhistidine is inserted in the amino acid position 49 in the A subunit,between wild-type amino acid positions 48 and
 49. 9. The CT-CRMaccording to claim 7, further comprising at least one additionalmutation in the A subunit of the cholera holotoxin at an amino acidposition other than the amino acid position 49 in the A subunit.
 10. TheCT-CRM according to claim 9, wherein said additional mutation is asubstitution for a subunit A amino acid selected from the groupconsisting of the arginine at amino acid position 7, the aspartic acidat amino acid position 9, the arginine at amino acid position 11, theglutamic acid at position 29, the histidine at amino acid position 44,the valine at amino acid position 53, the arginine at amino acidposition 54, the serine at amino acid position 61, the serine at aminoacid position 63, the histidine at amino acid position 70, the valine atamino acid position 97, the tyrosine at amino acid position 104, theproline at amino acid position 106, the histidine at amino acid position107, the serine at amino acid position 109, the glutamic acid at aminoacid position 110, the glutamic acid at amino acid position 112, theserine at amino acid position 114, the tryptophan at amino acid position127, the arginine at amino acid position 146, and the arginine at aminoacid position
 192. 11. The CT-CRM according to claim 9, wherein saidadditional mutation is selected from the group consisting of: a singleamino acid insertion in the amino acid position 25 in the A subunit, adouble amino acid insertion in the amino acid positions 35 and 36 in theA subunit, a single amino acid substitution in the amino acid position30, and a double amino acid insertion in the amino acid positions 31 and32 in the A subunit.
 12. An immunogenic, mutant cholera holotoxin(CT-CRM) comprising an amino acid sequence of subunit A of the wild-typecholera toxin (CT), wherein said subunit A comprises at least a doubleamino acid insertion in the amino acid positions 35 and 36 in the Asubunit, and wherein the mutant CT-CRM has reduced toxicity compared tosaid wild-type CT.
 13. The CT-CRM) according to claim 12, wherein theamino acids glycine and proline are inserted in the amino acid positions35 and 36 in the A subunit between wild-type amino acid positions 34 and35.
 14. The CT-CRM according to claim 12, further comprising at leastone additional mutation in the A subunit of the cholera holotoxin at anamino acid position other than the amino acid position 35 and 36 in theA subunit.
 15. The CT-CRM according to claim 14, wherein said additionalmutation is a substitution for a subunit A amino acid selected from thegroup consisting of the arginine at amino acid position 7, the asparticacid at amino acid position 9, the arginine at amino acid position 11,the glutamic acid at position 29, the histidine at amino acid position44, the valine at amino acid position 53, the arginine at amino acidposition 54, the serine at amino acid position 61, the serine at aminoacid position 63, the histidine at amino acid position 70, the valine atamino acid position 97, the tyrosine at amino acid position 104, theproline at amino acid position 106, the histidine at amino acid position107, the serine at amino acid position 109, the glutamic acid at aminoacid position 110, the glutamic acid at amino acid position 112, theserine at amino acid position 114, the tryptophan at amino acid position127, the arginine at amino acid position 146, and the arginine at aminoacid position
 192. 16. The CT-CRM according to claim 14, wherein saidadditional mutation is selected from the group consisting of a singleamino acid insertion in the amino acid position 25 in the A subunit, asingle amino acid insertion in the amino acid position 49 in the Asubunit, a single amino acid substitution in the amino acid position 30,and a double amino acid insertion in the amino acid positions 31 and 32in the A subunit.
 17. An immunogenic, mutant cholera holotoxin (CT-CRM)comprising an amino acid sequence of subunit A of the wild-type choleratoxin (CT), wherein said subunit A comprises at least a single aminoacid substitution in the amino acid position 30 and a double amino acidinsertion in the amino acid positions 31 and 32 in the A subunit, andwherein the mutant CT-CRM has reduced toxicity compared to saidwild-type CT.
 18. The CT-CRM according to claim 17, wherein the aminoacid tyrosine in the amino acid position 30 in the A subunit issubstituted with a tryptophan, and wherein amino acids alanine andhistidine are inserted in the amino acid positions 31 and 32 in the Asubunit between wild-type amino acid positions 30 and
 31. 19. The CT-CRMaccording to claim 17, further comprising at least one additionalmutation in the A subunit of the cholera holotoxin at an amino acidposition other than the amino acid positions 30, 31 and 32 in the Asubunit.
 20. The CT-CRM according to claim 19, wherein said additionalmutation is a substitution for a subunit A amino acid selected from thegroup consisting of the arginine at amino acid position 7, the asparticacid at amino acid position 9, the arginine at amino acid position 11,the glutamic acid at position 29, the histidine at amino acid position44, the valine at amino acid position 53, the arginine at amino acidposition 54, the serine at amino acid position 61, the serine at aminoacid position 63, the histidine at amino acid position 70, the valine atamino acid position 97, the tyrosine at amino acid position 104, theproline at amino acid position 106, the histidine at amino acid position107, the serine at amino acid position 109, the glutamic acid at aminoacid position 110, the glutamic acid at amino acid position 112, theserine at amino acid position 114, the tryptophan at amino acid position127, the arginine at amino acid position 146, and the arginine at aminoacid position
 192. 21. The CT-CRM according to claim 19, wherein saidadditional mutation is selected from the group consisting of: a singleamino acid insertion in the amino acid position 25 in the A subunit, asingle amino acid insertion in the amino acid position 49 in the Asubunit, and a double amino acid insertion in the amino acid positions35 and 36 in the A subunit.
 22. An immunogenic composition comprising amutant cholera holotoxin (CT-CRM) of any of claims 1 through 21, whereinthe mutant holotoxin enhances the immune response in a vertebrate hostto an antigen.
 23. The composition according to claim 22, wherein saidimmunogenic, mutant cholera holotoxin (CT-CRM) comprises an amino acidsequence of subunit A of the wild-type cholera toxin (CT), wherein theamino acid arginine in the amino acid position 25 in the A subunit issubstituted with a tryptophan.
 24. The composition according to claim22, wherein said immunogenic, mutant cholera holotoxin (CT-CRM)comprises an amino acid sequence of subunit A of the wild-type choleratoxin (CT), wherein the amino acid arginine in the amino acid position25 in the A subunit is substituted with a glycine.
 25. The compositionaccording to claim 22, wherein said immunogenic, mutant choleraholotoxin (CT-CRM) comprises an amino acid sequence of subunit A of thewild-type cholera toxin (CT), wherein the amino acid histidine isinserted in the amino acid position 49 in the A subunit, betweenwild-type amino acid positions 48 and
 49. 26. The composition accordingto claim 22, wherein said immunogenic, mutant cholera holotoxin (CT-CRM)comprises an amino acid sequence of subunit A of the wild-type choleratoxin (CT), wherein the amino acids glycine and proline are inserted inthe amino acid positions 35 and 36 in the A subunit between wild-typeamino acid positions 34 and
 35. 27. The composition according to claim22, wherein said immunogenic, mutant cholera holotoxin (CT-CRM)comprises an amino acid sequence of subunit A of the wild-type choleratoxin (CT), wherein the amino acid tyrosine in the amino acid position30 in the A subunit is substituted with a tryptophan, and wherein aminoacids alanine and histidine are inserted in the amino acid positions 31and 32 in the A subunit between wild-type amino acid positions 30 and31.
 28. The composition according to claim 22, further comprising anantigen derived from the member of the group consisting of a pathogenicbacterium, virus, fungus and parasite, a cancer cell, a tumor cell, anallergen and a self-molecule.
 29. The composition according to claim 28,wherein the selected bacterial antigen is a protein, polypeptide,peptide or fragment derived from a protein.
 30. The compositionaccording to claim 29, wherein the bacterial antigen is selected fromthe bacterial species consisting of typable and non-typable Haemophilusinfluenzae, Haemophilus somnus, Moraxella catarrhalis, Streptococcuspneumoniae, Streptococcus pyogenes, Streptococcus agalactiae,Streptococcus faecalis, Helicobacter pylori, Neisseria meningitidis,Neisseria gonorrhoeae, Chlamydia trachomatis, Chlamydia pneumoniae,Chlamydia psittaci, Bordetella pertussis, Alloiococcus otiditis,Salmonella typhi, Salmonella typhimurium, Salmonella choleraesuis,Escherichia coli, Shigella, Vibrio cholerae, Corynebacteriumdiptherieae, Mycobacterium tuberculosis, Mycobacteriumavium-Mycabacterium intracellulare complex, Proteus mirabilis, Proteusvulgaris, Staphylococcus aureus, Staphylococcus epidermidis, Clostridiumtetani, Leptospira interrogans, Borrelia burgdorferi, Pasteurellahaemolytica, Pasteurella multocida, Actinobacillus pleauropneumoniae andMycoplasma galliseptium.
 31. The composition according to claim 30,wherein the Haemophilus influenzae antigen is selected from the groupconsisting of the Haemophilus influenzae P4 outer membrane protein, theHaemophilus influenzae P6 outer membrane protein and Haemophilusinfluenzae adherence and penetration protein (Hap_(s)).
 32. Thecomposition according to claim 30, wherein the Helicobacter Pyloriantigen is the Helicobacter pylori urease protein.
 33. The compositionaccording to claim 30, wherein the Neissera meningitidis antigen isselected from the group consisting of the Neissera meningitidis Group Brecombinant class 1 pilin (rpilin) and the Neisseria meningitidis GroupB class 1 outer membrane protein (PorA).
 34. The composition accordingto claim 28, further comprising an antigen of a pathogenic virus. 35.The composition according to claim 28, wherein the selected viralantigen is a protein, polypeptide, peptide or fragment derived from aprotein.
 36. The composition according to claim 35, wherein the viralantigen is selected from the viral species consisting of Respiratorysyncytial virus, Parainfluenza virus types, 1,2,3, Humanmetapneumovirus, Influenza virus, Herpes simplex virus, Humancytomegalovirus, Human immunodeficiency virus, Hepatitis A virus,Hepatitis B virus, Hepatitis C virus, Human: papillomavirus, poliovirus,rotavirus, caliciviruses, measles virus, mumps virus, Rubella virus,adenovirus, rabies virus, canine distemper virus, rinderpest virus,avian pneumovirus, Hendra virus, Nipah virus, coronavirus, parvovirus,infectious rhinotracheitis viruses, feline leukemia virus, felineinfectious peritonitis virus, avian infectious bursal disease virus,Newcastle disease virus, Marek's disease virus, porcine respiratory andreproductive syndrome virus, equine arteritis virus and the encephalitisviruses.
 37. The composition according to claim 36, wherein therespiratory syncytial virus antigen is the respiratory syncytial virusfusion protein.
 38. The composition according to claim 36, wherein theherpes simplex virus (HSV) antigen is the herpes simplex virus (HSV)type 2 glycoprotein D (gD2).
 39. The composition according to claim 28,further comprising an antigen from a pathogenic fungus.
 40. Thecomposition according to claim 39, wherein the selected fungal antigenis a protein, polypeptide, peptide or fragment derived from a protein.41. The composition according to claim 40, wherein the fungal antigen isfrom a fungus selected from the group of pathogenic fungi consisting ofAspergillis, Blastomyces, Candida, Coccidiodes, Cryptococcus andHistoplasma.
 42. The composition according to claim 38, furthercomprising an antigen from a pathogenic parasite.
 43. The compositionaccording to claim 42, wherein the selected parasite antigen is aprotein, polypeptide, peptide or fragment derived from a protein. 44.The composition according to claim 42, wherein the parasite antigen isfrom a parasite selected from the group of pathogenic parasitesconsisting of Leishmania major, Ascaris, Trichuris, Giardia,Schistosoma, Cryptosporidium, Trichomonas, Toxoplasma gondii andPneumocystis carinii.
 45. The composition according to claim 28, whereinsaid antigen is derived from a cancer cell or tumor cell.
 46. Thecomposition according to claim 45, wherein said cancer or tumor cellantigen is selected from the group consisting of prostate specificantigen, carcino-embryonic antigen, MUC-1, Her2, CA-125, MAGE-3, ahormone, and a hormone analogs.
 47. The composition according to claim28, wherein said antigen is a polypeptide, peptide or fragment derivedfrom amyloid precursor protein, or an allergen.
 48. The compositionaccording to claim 47, wherein the amyloid precursor protein antigen isthe Aβ peptide, which is a 42 amino acid fragment of amyloid precursorprotein, or a fragment of the Aβ peptide.
 49. The composition accordingto claim 28, further comprising a diluent, excipient or carrier.
 50. Thecomposition according to claim 26, further comprising a second adjuvantin addition to the mutant cholera holotoxin.
 51. A method for enhancingthe immune response of a vertebrate host to an antigen, said methodcomprising administering to the host an immunogenic compositioncomprising a mutant cholera holotoxin (CT-CRM) of any of claims 1through 21, wherein the mutant holotoxin enhances the immune response ina vertebrate host to an antigen.
 52. An isolated and purified DNAsequence encoding an immunogenic, mutant cholera holotoxin of any ofclaims 1-21.
 53. An isolated and purified DNA encoding an immunogenic,mutant cholera holotoxin (CT-CRM) comprising a single amino acidsubstitution wherein the amino acid arginine in the amino acid position25 in the A subunit is substituted with a tryptophan.
 54. An isolatedand purified DNA encoding an immunogenic, mutant cholera holotoxin(CT-CRM) comprising a single amino acid substitution wherein the aminoacid arginine in the amino acid position 25 in the A subunit issubstituted with a glycine.
 55. An isolated and purified DNA encoding animmunogenic, mutant cholera holotoxin (CT-CRM) comprising a single aminoacid insertion wherein the amino acid residue histidine is inserted inthe amino acid position 49 in the A subunit between wild-type amino acidpositions 48 and
 49. 56. An isolated and purified DNA encoding animmunogenic, mutant cholera holotoxin (CT-CRM) comprising a double aminoacid insertion wherein the amino acid residues glycine and proline areinserted in the amino acid positions 35 and 36 in the A subunit betweenwild-type amino acid positions 34 and
 35. 57. An isolated and purifiedDNA encoding a immunogenic, mutant cholera holotoxin (CT-CRM) comprisinga single amino acid substitution and a double amino acid insertionwherein the amino acid tyrosine in the amino acid position 30 in the Asubunit is substituted with a tryptophan, and wherein amino acidsalanine and histidine are inserted in the amino acid positions 31 and 32in the A subunit between wild-type amino acid positions 30 and
 31. 58. Anucleic acid molecule comprising an isolated and purified nucleic acidsequence encoding an immunogenic, mutant cholera holotoxin of any ofclaims 1-21, wherein the sequence encoding the immunogenic, mutantcholera holotoxin is operatively linked to regulatory sequences enablingexpression of said mutant holotoxin in a host cell.
 59. The moleculeaccording to claim 58, wherein said regulatory sequence is an induciblepromoter.
 60. The molecule according to claim 50, wherein said promoteris the arabinose inducible promoter.
 61. The molecule according to claim58, wherein said molecule is a viral or non-viral vector.
 62. Themolecule according to claim 61, wherein said non-viral vector is a DNAplasmid.
 63. A host cell transformed, transduced, infected ortransfected with a nucleic acid molecule comprising an isolated andpurified nucleic acid sequence encoding an immunogenic, mutant choleraholotoxin of any of claims: 1-21, wherein the sequence encoding theimmunogenic, mutant cholera holotoxin is operatively linked toregulatory sequences enabling expression of said mutant holotoxin in ahost cell.
 64. A method of producing an immunogenic mutant choleraholotoxin, wherein the cholera holotoxin has reduced toxicity comparedto a wild-type cholera holotoxin comprising culturing a host celltransformed, transduced, infected or transfected with a nucleic acidmolecule comprising an isolated and purified nucleic acid sequenceencoding an immunogenic, mutant cholera holotoxin of any of claims 1-21,wherein the sequence encoding the immunogenic, mutant cholera holotoxinis operatively linked to regulatory sequences enabling expression ofsaid mutant holotoxin in a host cell under conditions which permit theexpression of said immunogenic mutant cholera holotoxin by the hostcell.
 65. Use of an effective adjuvanting amount of a mutant choleraholotoxin of any of claims 1 through 21, in combination with a selectedantigen from a pathogenic bacterium, virus, fungus, parasite, a cancercell, a tumor cell, an allergen, a self molecule, or vertebrate antigento prepare an antigenic composition, wherein said mutant holotoxinenhances the immune response in a vertebrate host to said antigenvertebrate host to said antigen.